Hypersensitive response elicitor fragments eliciting a hypersensitive response and uses thereof

ABSTRACT

The present invention is directed to isolated fragments of an Erwinia hypersensitive response elicitor protein or polypeptide which fragments elicit a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode the Erwinia hypersensitive response eliciting fragment. Isolated fragments of hypersensitive response elicitor proteins or polypeptides, which elicit a hypersensitive response, and the isolated DNA molecules that encode them can used to impart disease resistance to plants, to enhance plant growth, and/or to control insects on plants. This can be achieved by applying the hypersensitive response eliciting fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding a hypersensitive response eliciting fragment can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.

This application claims benefit of U.S. patent application Ser. No.60/048,109, filed May 30, 1997.

FIELD OF THE INVENTION

The present invention relates to fragments of a hypersensitive responseelicitor which fragments elicit a hypersensitive response and usesthereof.

BACKGROUND OF THE INVENTION

Interactions between bacterial pathogens and their plant hosts generallyfall into two categories: (1) compatible (pathogen-host), leading tointercellular bacterial growth, symptom development, and diseasedevelopment in the host plant; and (2) incompatible (pathogen-nonhost),resulting in the hypersensitive response, a particular type ofincompatible interaction occurring, without progressive diseasesymptoms. During compatible interactions on host plants, bacterialpopulations increase dramatically and progressive symptoms occur. Duringincompatible interactions, bacterial populations do not increase, andprogressive symptoms do not occur.

The hypersensitive response is a rapid, localized necrosis that isassociated with the active defense of plants against many pathogens(Kiraly, Z., “Defenses Triggered by the Invader: Hypersensitivity,”pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J. G.Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement,Z., “Hypersensitivity,” pages 149-177 in: Phytopathogenic Prokaryotes,Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York(1982)). The hypersensitive response elicited by bacteria is readilyobserved as a tissue collapse if high concentrations (≧10⁷ cells/ml) ofa limited host-range pathogen like Pseudonmonas syringae or Erwiniaamylovora are infiltrated into the leaves of nonhost plants (necrosisoccurs only in isolated plant cells at lower levels of inoculum)(Klement, Z. “Rapid Detection of Pathogenicity of PhytopathogenicPseudomonads,” Nature 199:299-300; Klement, et al., “HypersensitiveReaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,”Phytopathology 54:474-477 (1963); Turner, et al., “The QuantitativeRelation Between Plant and Bacterial Cells Involved in theHypersensitive Reaction,” Phytopathology 64:885-890 (1974); Klement, Z.,“Hypersensitivity,” pages 149-177 in Phytopathoenic Prokaryotes, Vol.2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)).The capacities to elicit the hypersensitive response in a nonhost and bepathogenic in a host appear linked. As noted by Klement, Z.,“Hypersensitivity,” pages 149-177 in Phytopathogenic Prokaryotes, Vol.2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York, thesepathogens also cause physiologically similar, albeit delayed, necrosesin their interactions with compatible hosts. Furthermore, the ability toproduce the hypersensitive response or pathogenesis is dependent on acommon set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Clusterof Pseudomonas syringae pv. ‘phaseolicola’ Controls Pathogenicity ofBean Plants and Hypersensitivity on Nonhost Plants,” J. Bacteriol.168:512-22 (1986); Willis, D. K., et al., “hrp Genes of PhytopathogenicBacteria,” Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently,the hypersensitive response may hold clues to both the nature of plantdefense and the basis for bacterial pathogenicity.

The hrp genes are widespread in gram-negative plant pathogens, wherethey are clustered, conserved, and in some cases interchangeable(Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol.Plant-Microbe Interact. 4:132-138 (1991); Bonas, U., “hrp Genes ofPhytopathogenic Bacteria,” pages 79-98 in: Current Topics inMicrobiology and Immunology: Bacterial Pathogenesis of Plants andAnimals—Molecular and Cellular Mechanisms, J. L. Dangl, ed.Springer-Verlag, Berlin (1994)). Several hrp genes encode components ofa protein secretion pathway similar to one used by Yersinia, Shigella,and Salmonella spp. to secrete proteins essential in animal diseases(Van Gijsegem, et al., “Evolutionary Conservation of PathogenicityDeterminants Among Plant and Animal Pathogenic Bacteria,” TrendsMicrobiol. 1:175-180 (1993)). In E. amylovora, P. syringae, and P.solanacearum, hrp genes have been shown to control the production andsecretion of glycine-rich, protein elicitors of the hypersensitiveresponse (He, S. Y., et al. “Pseudomonas Syringae pv. SyringaeHarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicitsthe Hypersensitive Response in Plants,” Cell 73:1255-1266 (1993), Wei,Z.-H., et al., “HrpI of Erwinia amylovora Functions in Secretion ofHarpin and is a Member of a New Protein Family,” J. Bacteriol.175:7958-7967 (1993); Arlat, M. et al. “PopA1, a Protein Which Induces aHypersensitive-like Response on Specific Petunia Genotypes, is Secretedvia the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543-553(1994)).

The first of these proteins was discovered in E. amylovora Ea321, abacterium that causes fire blight of rosaceous plants, and wasdesignated harpin (Wei, Z.-M., et al, “Harpin, Elicitor of theHypersensitive Response Produced by the Plant Pathogen Erwiniaamylovora,” Science 257:85-88 (1992)). Mutations in the encoding hrpNgene revealed that harpin is required for E. amylovora to elicit ahypersensitive response in nonhost tobacco leaves and incite diseasesymptoms in highly susceptible pear fruit. The P. solanacearum GMI1000PopA1 protein has similar physical properties and also elicits thehypersensitive response in leaves of tobacco, which is not a host ofthat strain (Arlat, et al. “PopA1, a Protein Which Induces aHypersensitive-like Response on Specific Petunia Genotypes, is Secretedvia the Hrp Pathway of Pseudomnonas solanacearum,” EMBO J. 13:543-53(1994)). However, P. solanacearum popA mutants still elicit thehypersensitive response in tobacco and incite disease in tomato. Thus,the role of these glycine-rich hypersensitive response elicitors canvary widely among gram-negative plant pathogens.

Other plant pathogenic hypersensitive response elicitors have beenisolated, cloned, and sequenced. These include: Erwinia chrysanthemi(Bauer, et. al., “Erwinia chrysanthemi Harpin_(Ech): Soft-RotPathogenesis,” MPMI 8(4): 484-91 (1995)); Erwinia carotovora (Cui, et.al., “The RsmA⁻ Mutants of Erwinia carotovora subsp. carotovora StrainEcc71 Overexpress hrpN_(Ecc) and Elicit a Hypersensitive Reaction-likeResponse in Tobacco Leaves,” MPMI 9(7): 565-73 (1966)); Erwiniastewartii (Ahmad, et. al., “Harpin is not Necessary for thePathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec.Plant-Microb. Inter. Jul. 14-19, 1996 and Ahmad, et. al., “Harpin is notNecessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann.Mtg. Am. Phytopath. Soc. Jul. 27-31, 1996); and Pseudomonas syringae pv.syringae (WO 94/26782 to Cornell Research Foundation, Inc.).

The present invention seeks to identify fragments of hypersensitiveresponse elicitor proteins or polypeptides, which fragments elicit ahypersensitive response, and uses of such fragments.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated fragment of an Erwiniahypersensitive response elicitor protein or polypeptide where thefragment elicits a hypersensitive response in plants. Also disclosed areisolated DNA molecules which encode such fragments.

The fragments of hypersensitive response elicitors can be used to impartdisease resistance to plants, to enhance plant growth, and/or to controlinsects. This involves applying the fragments in a non-infectious formto plants or plant seeds under conditions effective to impart diseaseresistance, to enhance plant growth, and/or to control insects on plantsor plants grown from the plant seeds.

As an alternative to applying the fragments to plants or plant seeds inorder to impart disease resistance, to enhance plant growth, and/or tocontrol insects on plants, transgenic plants or plant seeds can beutilized. When utilizing transgenic plants, this involves providing atransgenic plant transformed with a DNA molecule encoding a fragment ofa hypersensitive response elicitor protein or polypeptide whichfragments elicit a hypersensitive response in plants and growing theplant under conditions effective to impart disease resistance, toenhance plant growth, and/or to control insects in the plants or plantsgrown from the plant seeds. Alternatively, a transgenic plant seedtransformed with the DNA molecule encoding such a fragment can beprovided and planted in soil. A plant is then propagated underconditions effective to impart disease resistance, to enhance plantgrowth, and/or to control insects on plants or plants grown from theplant seeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a deletion and proteolysis analysis for the Erwiniaamylovora hypersensitive response elicitor (i.e. harpin). A is the nameof the harpin fragment. B is the length of the fragment in amino acidresidues. C indicates whether detectable protein is produced. D stateswhether there is hypersensitive response (i.e., HR) eliciting activity.The solid line indicates that there are additional amino acids which arenot harpin encoded, while the dashed line indicates the portion of theharpin that is deleted. The numbers above the fragments in the boxrepresent the amino acid residue present at the end of a given fragment;residue #1 is the N-terminus, and residue #403 is the C-terminus.

FIG. 2 is a Western blot illustrating specific secretion of harpin_(Ea),but not harpin_(Ea)C31. Lane A, Ea273(pGP1-2) CFEP; lane B,Ea273(pGP1-2)(pCPP1104) CFEP; lane C, E. coli DH5α (pCPP1107) CFEPharpin size standard; lane D, BioRad low range molecular weight markers;lane E, Ea273(pGP1-2) supernatant; lane F, Ea273(pGP1 2)(pCPP 1104)supernatant. The blot was probed with an anti-harpin_(Ea) polyclonalantibody.

FIG. 3 is an HR assay on tobacco leaf infiltrated as follows: (1) A,harpin_(Ea)+raspberry IF; (2) B, harpin_(Ea)+apple IF; (3) C,harpin_(Ea)+tobacco IF; (4) D, harpin_(Ea)+endoproteinase Glu-C; (5) E,harpin_(Ea)+trypsin; (6) F, harpin_(Ea); (7) G, tobacco IF; (8) H.endoproteinase Glu-C; (9) I, trypsin; and (10) J, harpin_(Ea). IF refersto intracelluar fluids.

FIG. 4 shows the digestion of harpin with endoproteinase Glu-C. Lane Ais harpin; Lane B is harpin+endoproteinase Glu-C; Lane C is BioRad lowrange molecular weight markers.

FIG. 5A shows the proteolysis of harpin. Coomassie blue stainedpolyacrylamide gel was loaded as follows: A, BioRad low range molecularweight markers; B, IF-apple; C, IF-raspberry; D, IF-tobacco; E,harpin_(Ea); F, harpin_(Ea)+IF-apple; G, harpin_(Ea)+IF-raspberry; H,harpin_(Ea)+IF-tobacco.

FIG. 5B shows a Coomassie Blue stained polyacrylamide gel loaded asfollows: A, IF-tobacco; B, IF-tobacco+harpin_(Ea); C, harpin_(Ea); D,BioRad low range molecular weight markers; E,IF-tobacco+harpin_(Ea)+PMSF. HR-eliciting activity of the samplefollowing proteolysis is denoted below the gel.

FIG. 5C depicts whether proteolytic activity is present in IF from allplants tested. Intercellular fluid harvested from several plants wasanalyzed by PAGE in a gel containing 0.1% copolymerized gelatin. Afterwashing to remove SDS and incubation to allow proteolysis of gelatin,the gels were stained to demonstrate the presence of gelatinolyticactivity. A, IF-apple; B, IF-tobacco; C, IF-cotoneaster; D, BioRad mw;E, endoproteinase Glu-C; and F, ground leaf extract-tobacco.

FIG. 6 shows the refractionation of elicitor-active peptides followingproteolysis of harpin_(Ea) by tobacco IF. Absorbance was measured at 210nm. Peak 1 contains peptides P91 and P95; peak 2 contains peptides P65and P69.

FIG. 7 shows the predicted proteolytic cleavage sites within harpin ofseveral tested proteinases, and the effect of these cleavages onactivity of active harpin fragments. Residues potentially important forHR-eliciting activity, based on the loss of activity following furthercleavage, are indicated by upward-pointing arrows at bottom.

FIG. 8 shows the similarities near N-termini among harpins of Erwiniaspp. Underlined residues are present (identical or similar) in at leastfour out of the five proteins examined. Nine out of the first 26residues are conserved in this manner.

FIGS. 9A and 9B show Kyte-Doolittle hydropathy plots of bacterialHR-eliciting proteins. Ea, E. amylovora EA321; Est, E. stewartii DC283;Ech, E. chrysanthemi AC4150; Ecc, E. cartovora subsp. carotovora; Rs, R,solanacearum; Pss, P. syringae pv. syringae.

FIG. 10 shows truncated proteins of the hypersensitive response elicitorprotein or polypeptide.

FIG. 11 shows a list of synthesized oligonucleotide primers forconstruction of truncated harpin proteins. N represents the N-terminus(5′ region), and C represents the C-terminus (3′ region). The primerscorrespond to the indicated sequence identification numbers for thepresent application: N1 (SEQ. ID. No. 1), N76 (SEQ. ID. No.2), N99 (SEQ.ID. No.3), N105 (SEQ. ID. No.4), N 110 (SEQ. ID. No.5), N137 (SEQ. ID.No.6), N150 (SEQ. ID. No.7), N169 (SEQ. ID. No.8), N210 (SEQ. ID. No.9), N267 (SEQ. ID. No. 10), N343 (SEQ. ID. No. 11), C75 (SEQ. ID.No.12), C104 (SEQ. ID. No.13), C168 (SEQ. ID. No.14), C180 (SEQ. ID. No.15), C204 (SEQ. ID. No.16), C209 (SEQ. ID. No. 17), C266 (SEQ. ID. No.18), C342 (SEQ. ID. No.19), and C403 (SEQ. ID. No.20).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to isolated fragments of ahypersensitive response elicitor protein or polypeptide where thefragments elicit a hypersensitive response in plants. Also disclosed areDNA molecules encoding such fragments as well as expression systems,host cells, and plants containing such molecules. Uses of the fragmentsthemselves and the DNA molecules encoding them are disclosed.

The fragments of hypersensitive response elicitor polypeptides orproteins according to the present invention are derived fromhypersensitive response elicitor polypeptides or proteins of a widevariety of fungal and bacterial pathogens. Such polypeptides or proteinsare able to elicit local necrosis in plant tissue contacted by theelicitor. Examples of suitable bacterial sources of polypeptide orprotein elicitors include Erwinia, Pseudomonas, and Xanthamonas species(e.g., the following bacteria: Erwinia amylovora, Erwinia chrysanthemi,Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomassolancearum, Xanthomonas campestris, and mixtures thereof).

An example of a fungal source of a hypersensitive response elicitorprotein or polypeptide is Phytophthora. Suitable species of Phytophthorainclude Phytophthora parasitica, Phytophthora cryptogea, Phytophthoracinnamomi, Phytophthora capsici, Phytophthora megasperma, andPhytophthora citrophthora.

The hypersensitive response elicitor polypeptide or protein from Erwiniachrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 21as follows:

Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser1                5               10               15 Gly Leu Gly Ala GlnGly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser            20                  25                  30 Leu Gly Ser SerVal Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr        35                  40                  45 Ser Ala Leu Thr SerMet Met Phe Gly Gly Ala Leu Ala Gln Gly Leu    50                  55                  60 Gly Ala Ser Ser Lys GlyLeu Gly Met Ser Asn Gln Leu Gly Gln Ser65                  70                  75                  80 Phe GlyAsn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys                85                  90                  95 Ser Gly GlyAsp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp            100                 105                  110 Leu Leu Gly HisAsp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln        115                 120                 125 Leu Ala Asn Ser MetLeu Asn Ala Ser Gln Met Thr Gln Gly Asn Met    130                 135                 140 Asn Ala Phe Gly Ser GlyVal Asn Asn Ala Leu Ser Ser Ile Leu Gly145                 150                 155                 160 Asn GlyLeu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly                165                 170                 175 Ala Gly GlyLeu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu            180                 185                 190 Gly Asn Ala IleGly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala        195                 200                 205 Leu Ser Asn Val SerThr His Val Asp Gly Asn Asn Arg His Phe Val    210                 215                 220 Asp Lys Glu Asp Arg GlyMet Ala Lys Glu Ile Gly Gln Phe Met Asp225                 230                 235                 240 Gln TyrPro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp                245                 250                 255 Ser Ser ProLys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys            260                 265                 270 Pro Asp Asp AspGly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln        275                 280                 285 Ala Met Gly Met IleLys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr    290                 295                 300 Asn Leu Asn Leu Arg GlyAla Gly Gly Ala Ser Leu Gly Ile Asp Ala305                 310                 315                 320 Ala ValVal Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala                325                 330                 335 Asn Ala

This hypersensitive response elicitor polypeptide or protein has amolecular weight of 34 kDa, is heat stable, has a glycine content ofgreater than 16%, and contains substantially no cysteine. The Erwinachrysanthemi hypersensitive response elicitor polypeptide or protein isencoded by a DNA molecule having a nucleotide sequence corresponding toSEQ. ID. No. 22 as follows:

CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CACCGTTACG   60GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC  120GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG  180CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG  240TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG  300CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GGCGGGAATG  360ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CGGACGCGCC  420CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GGAACCGTTT  480CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG  540GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA  600AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC  660TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TGGATAAACT  720GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GCGGCGCGCT  780GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TGGGCCAGTC  840TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA  900TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC  960CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC 1020CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CCATTCTCGG 1080CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CAGGCGGCTT 1140GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA 1380GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GTATGACCGG 1440CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TGGCGGGTGA 1500TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA 1740ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CATCTTCCTC 1800GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA ACTCGCCGGC 1860CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141

The hypersentive response elicitor polypeptide or protein derived fromErwina amylovora has an amino acid sequence corresponding to SEQ. ID.No. 23 as follows:

Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gin Ile Ser1               5                   10                  15 Ile Gly GlyAla Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln            20                  25                  30 Asn Ala Gly LeuGly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn        35                  40                  45 Gln Asn Asp Thr ValAsn Gln Leu Ala Gly Leu Leu Thr Gly Met Met    50                  55                  60 Met Met Met Ser Met MetGly Gly Gly Gly Leu Met Gly Gly Gly Leu65                  70                  75                  80 Gly GlyGly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu                85                  90                  95 Gly Leu SerAsn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr            100                 105                 110 Leu Gly Ser LysGly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro        115                 120                 125 Leu Asp Gln Ala LeuGly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser    130                 135                 140 Thr Ser Gly Thr Asp SerThr Ser Asp Ser Ser Asp Pro Met Gln Gln 145                150                 155                 160 Leu Leu LysMet Phe Ser Glu Ile Met Gln Ser Leu Phe Gly Asp Gly                165                 170                 175 Gln Asp GlyThr Gln Gly Ser Ser Ser Gly Gly Lys Gln Pro Thr Glu            180                 185                 190 Gly Glu Gln AsnAla Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly        195                 200                 205 Leu Met Gly Asn GlyLeu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly    210                 215                 220 Gly Gly Gln Gly Gly AsnAla Gly Thr Gly Leu Asp Gly Ser Ser Leu225                 230                 235                 240 Gly GlyLys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln                245                 250                 255 Leu Gly AsnAla Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln            260                 265                 270 Ala Leu Asn AspIle Gly Thr His Arg His Ser Ser Thr Arg Ser Phe        275                 280                 285 Val Asn Lys Gly AspArg Ala Met Ala Lys Glu Ile Gly Gln Phe Met    290                 295                 300 Asp Gln Tyr Pro Glu ValPhe Gly Lys Pro Gln Tyr Gln Lys Gly Pro305                 310                 315                 320 Gly GlnGlu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser                325                 330                 335 Lys Pro AspAsp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn            340                 345                 350 Lys Ala Lys GlyMet Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn        355                 360                 365 Gly Asn Leu Gln AlaArg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp    370                 375                 380 Ala Met Met Ala Gly AspAla Ile Asn Asn Met Ala Leu Gly Lys Leu385                 390                 395                 400 Gly AlaAla

This hypersensitive response elicitor polypeptide or protein has amolecular weight of about 39 kDa, has a pI of approximately 4.3, and isheat stable at 100° C. for at least 10 minutes. This hypersensitiveresponse elicitor polypeptide or protein has substantially no cystcine.The hypersensitive response elicitor polypeptide or protein derived fromErwinia amylovora is more fully described in Wei, Z.-M., R. J. Laby, C.H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V. Beer, “Harpin,Elicitor of the Hypersensitive Response Produced by the Plant PathogenErwinia amylovora,” Science 257:85-88 (1992), which is herebyincorporated by reference. The DNA molecule encoding this polypeptide orprotein has a nucleotide sequence corresponding to SEQ. ID. No. 24 asfollows:

AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA   60GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GCAAATTTCT  120ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG  180GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG  240GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG  300GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA  360GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA  420GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GGGTATTAAC  480TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC  540CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG  600CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC  660GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG  720CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC  780GGTTCCTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CTACCAGCAG  840TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT  900ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG  960GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC 1140ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC ACGCGCTGCC 1200GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288

The hypersensitive response elicitor polypeptide or protein derived fromPseudomonas syringae has an amino acid sequence corresponding to SEQ.ID. No. 25 as follows:

Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met1               5                   10                  15 Ala Leu ValLeu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser            20                  25                  30 Ser Lys Ala LeuGln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met        35                  40                  45 Arg Asn Gly Gln LeuAsp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala    50                  55                  60 Lys Ser Met Ala Ala AspGly Lys Ala Gly Gly Gly Ile Glu Asp Val65                  70                  75                  80 Ile AlaAla Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe                85                  90                  95 Gly Ala SerAla Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met            100                 105                 110 Thr Gln Val LeuAsn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu        115                 120                 125 Thr Lys Gln Asp GlyGly Thr Ser Phe Ser Glu Asp Asp Met Pro Met    130                 135                 140 Leu Asn Lys Ile Ala GlnPhe Met Asp Asp Asn Pro Ala Gln Phe Pro145                 150                 155                 160 Lys ProAsp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe                165                 170                 175 Leu Asp GlyAsp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile            180                 185                 190 Gly Gln Gln LeuGly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly        195                 200                 205 Thr Gly Gly Gly LeuGly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser    210                 215                 220 Val Met Gly Asp Pro LeuIle Asp Ala Asn Thr Gly Pro Gly Asp Ser225                 230                 235                 240 Gly AsnThr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp                245                 250                 255 Arg Gly LeuGln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val            260                 265                 270 Asn Thr Pro GlnThr Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala Gln         275                280                 285 Asp Leu Asp Gln Leu Leu Gly GlyLeu Leu Leu Lys Gly Leu Glu Ala    290                 295                 300 Thr Leu Lys Asp Ala GlyGln Thr Gly Thr Asp Val Gln Ser Ser Ala305                 310                 315                 320 Ala GlnIle Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg                325                 330                 335 Asn Gln AlaAla Ala             340

This hypersensitive response elicitor polypeptide or protein has amolecular weight of 34-35 kDa. It is rich in glycine (about 13.5%) andlacks cysteine and tyrosine. Further information about thehypersensitive response elicitor derived from Pseudomonas syringae isfound in He, S. Y., H. C. Huang, and A. Collmer, “Pseudomonas syringaepv. syringae Harpin_(Pss): a Protein that is Secreted via the HrpPathway and Elicits the Hypersensitive Response in Plants,” Cell73:1255-1266 (1993), which is hereby incorporated by reference. The DNAmolecule encoding the hypersensitive response elicitor from Pseudomonassyringae has a nucleotide sequence corresponding to SEQ. ID. No. 26 asfollows:

ATGCACAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CCTTCTCCTG   60GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC  120GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA  180AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC  240ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG  300GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TGGCCTGGCC  360AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CTCCGAAGAC  420GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC ACAGTTTCCC  480AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC  540GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG  600AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC  660AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC  720GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TGGCCTGCAA  780TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CGGTACGTCG  840GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG  900GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT  960GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA 1020GCCTGA 1026

The hypersensitive response elicitor polypeptide or protein derived fromPseudomonas solanacearum has an amino acid sequence corresponding toSEQ. ID. No. 27 as follows:

Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln1               5                   10                  15 Asn Leu AsnLeu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser            20                  25                  30 Val Gln Asp LeuIle Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile        35                  40                  45 Ala Ala Leu Val GlnLys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly    50                  55                  60 Asn Thr Gly Asn Ala ProAla Lys Asp Gly Asn Ala Asn Ala Gly Ala65                  70                  75                  80 Asn AspPro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser                85                  90                  95 Ala Asn LysThr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met            100                 105                 110 Gln Ala Leu MetGln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala        115                 120                 125 Ala Leu His Met GlnGln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val    130                 135                 140 Gly Gly Ala Asn Gly AlaLys Gly Ala Gly Gly Gln Gly Gly Leu Ala145                 150                 155                 160 Glu AlaLeu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly                165                 170                 175 Gly Ala GlyAla Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly            180                 185                 190 Ala Asp Gly GlySer Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala        195                 200                 205 Asp Gly Gly Asn GlyVal Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn    210                 215                 220 Ala Gly Asp Val Asn GlyAla Asn Gly Ala Asp Asp Gly Ser Glu Asp225                 230                 235                 240 Gln GlyGly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn                245                 250                 255 Ala Leu ValGln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln            260                 265                 270 Ala Gln Gly GlySer Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly        275                 280                 285 Ala Asn Pro Gly AlaAsn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser    290                 295                 300 Gly Gln Asn Asn Leu GlnSer Gin Ile Met Asp Val Val Lys Glu Val305                 310                 315                 320 Val GlnIle Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln                325                 330                 335 Gln Ser ThrSer Thr Gln Pro Met             340

It is encoded by a DNA molecule having a nucleotide sequencecorresponding SEQ. ID. No. 28 as follows:

ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CCTGAACCTC   60AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CAAGCAGGTC  120GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC  180GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC  240AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC  300GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GCTGCTGGAA  360GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CAATGACAAG  420GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC  480GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC  540GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT  600GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC  660GGCCCGCAGA ACGCAGGCGA TGTCAACCCT GCCAACGGCC CGGATGACGG CAGCGAAGAC  720CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC GCTGGTGCAG  780ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC AGGGCGGCTC GAAGGGTGCC  840GGCAACGCCT CGCCGGCTTC CGCCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT  900GATCAATCGT CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC  960GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020ACGCAGCCGA TGTAA 1035

Further information regarding the hypersensitive response elicitorpolypeptide or protein derived from Pseudomonas solanacearum is setforth in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet, and C.A. Boucher, “PopA1, a Protein which Induces a Hypersensitive-likeResponse in Specific Petunia Genotypes, is Secreted via the Hrp Pathwayof Pseudomonas solanacearun,” EMBO J. 13:543-533 (1994), which is herebyincorporated by reference.

The hypersensitive response elicitor polypeptide or protein fromXanthomonas campestris pv. glycines has an amino acid sequencecorresponding to SEQ. ID. No. 29 as follows:

Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile1               5                   10    Ala Ile Leu Ala Ala Ile AlaLeu Pro Ala Tyr Gln         15                  20 Asp Tyr 25

This sequence is an amino terminal sequence having only 26 residues fromthe hypersensitive response elicitor polypeptide or protein ofXanthomonas campestris pv. glycines. It matches with fimbrial subunitproteins determined in other Xanthomonas campestris pathovars.

The hypersensitive response elicitor polypeptide or protein fromXanthomonas cainpestris pv. pelargonii is heat stable, proteasesensitive, and has a molecular weight of 20 kDa. It includes an aminoacid sequence corresponding to SEQ. ID. No. 30 as follows:

Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln1               5                   10 Gln Leu Asp Gln Leu Leu Ala Met        15                  20

Isolation of Erwinia carotovora hypersensitive response elictor proteinor polypeptide is described in Cui et al., “The RsmA Mutants of Erwiniacarotovora subsp. carotovora Strain Ecc71 Overexpress hrp N_(Ecc), andElicit a Hypersensitive Reaction-like Response in Tobacco Leaves,” MPMI,9(7):565-73 (1996), which is hereby incorporated by reference. Thehypersensitive response elicitor protein or polypeptide of Erwiniastewartii is set forth in Ahmad et al., “Harpin is Not Necessary for thePathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec.Plant-Microbe Interact., July 14-19, 1996 and Ahmad, et al., “Harpin isNot Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann.Mtg. Am. Phytopath. Soc., Jul. 27-31, 1996, which are herebyincorporated by reference.

Hypersensitive response elicitor proteins or polypeptides fromPhytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamoni,Phytophthora capsici, Phytophthora megasperma, and Phytophoracitrophthora are described in Kaman, et al., “Extracellular ProteinElicitors from Phytophthora: Most Specificity and Induction ofResistance to Bacterial and Fungal Phytopathogens,” Molec. Plant-MicrobeInteract., 6(1):15-25 (1993), Ricci et al., “Structure and Activity ofProteins from Pathogenic Fungi Phytophthora Eliciting Necrosis andAcquired Resistance in Tobacco,” Eur. J. Biochem., 183:555-63 (1989),Ricci et al., “Differential Production of Parasiticein, and Elicitor ofNecrosis and Resistance in Tobacco, by Isolates of Phytophthoraparasitica,” Plant Path. 41:298-307 (1992), Baillreul et al, “A NewElicitor of the Hypersensitive Response in Tobacco: A FungalGlycoprotein Elicits Cell Death, Expression of Defence Genes, Productionof Salicylic Acid, and Induction of Systemic Acquired Resistance,” PlantJ., 8(4):551-60 (1995), and Bonnet et al., “Acquired ResistanceTriggered by Elicitors in Tobacco and Other Plants,” Eur. J. PlantPath., 102:181-92 (1996), which are hereby incorporated by reference.

The above elicitors are exemplary. Other elicitors can be identified bygrowing fungi or bacteria that elicit a hypersensitive response underwhich genes encoding an elicitor are expressed. Cell-free preparationsfrom culture supernatants can be tested for elicitor activity (i.e.local necrosis) by using them to infiltrate appropriate plant tissues.

Fragments of the above hypersensitive response elicitor polypeptides orproteins as well as fragments of full length elicitors from otherpathogens are encompassed by the method of the present invention.

Suitable fragments can be produced by several means. In the first,subclones of the gene encoding a known elicitor protein are produced byconventional molecular genetic manipulation by subcloning genefragments. The subclones then are expressed in vitro or in vivo inbacterial cells to yield a smaller protein or peptide that can be testedfor elicitor activity according to the procedure described below.

As an alternative, fragments of an elicitor protein can be produced bydigestion of a full-length elicitor protein with proteolytic enzymeslike chymotrypsin or Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave elicitor proteins at differentsites based on the amino acid sequence of the elicitor protein. Some ofthe fragments that result from proteolysis may be active elicitors ofresistance.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the elicitor protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for expression of a truncated peptide orprotein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theelicitor being produced. Alternatively, subjecting a full lengthelicitor to high temperatures and pressures will produce fragments.These fragments can then be separated by conventional procedures (e.g.,chromatography, SDS-PAGE).

An example of suitable fragments of an Erwinia hypersensitive responseelicitor which fragments elicit a hypersensitive response are fragmentsof the Erwinia amylovora hypersensitive response elicitor. Suitablefragments include a C-terminal fragment of the amino acid sequence ofSEQ. ID. No. 23, an N-terminal fragment of the amino acid sequence ofSEQ. ID. No. 23, or an internal fragment of the amino acid sequence ofSEQ. ID. No. 23. The C-terminal fragment of the amino acid sequence ofSEQ. ID. No. 23 can span amino acids 105 and 403 of SEQ. ID. No. 23. TheN-terminal fragment of the amino acid sequence of SEQ. ID. No. 23 canspan the following amino acids of SEQ. ID. No. 23: 1 and 98, 1 and 104,1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1and 372. The internal fragment of the amino acid sequence of SEQ. ID.No. 23 can span the following amino acids of SEQ. ID. No. 23: 76 and209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109and 200, 137 and 180, and 105 and 180. Other suitable fragments can beidentified in accordance with the present invention.

Variants may be made by, for example, the deletion or addition of aminoacids that have minimal influence on the properties, secondary structureand hydropathic nature of the polypeptide. For example, a polypeptidemay be conjugated to a signal (or leader) sequence at the N-terminal endof the protein which co-translationally or post-translationally directstransfer of the protein. The polypeptide may also be conjugated to alinker or other sequence for ease of synthesis, purification, oridentification of the polypeptide.

The fragment of the present invention is preferably produced in purifiedform (preferably at least about 60%, more preferably 80%, pure) byconventional techniques. Typically, the fragment of the presentinvention is produced but not secreted into the growth medium ofrecombinant host cells. Alternatively, the protein or polypeptide of thepresent invention is secreted into growth medium. In the case ofunsecreted protein, to isolate the protein fragment, the host cell(e.g., E. coli) carrying a recombinant plasmid is propagated, lysed bysonication, heat, or chemical treatment, and the homogenate iscentrifuged to remove bacterial debris. The supernatant is thensubjected to heat treatment and the fragment is separated bycentrifugation. The supernatant fraction containing the fragment issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the fragment. If necessary, theprotein fraction may be further purified by ion exchange or HPLC.

The DNA molecule encoding the fragment of the hypersensitive responseelicitor polypeptide or protein can be incorporated in cells usingconventional recombinant DNA technology. Generally, this involvesinserting the DNA molecule into an expression system to which the DNAmolecule is heterologous (i.e. not normally present). The heterologousDNA molecule is inserted into the expression system or vector in propersense orientation and correct reading frame. The vector contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinavirus. Recombinant viruses can be generated by transection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated byreference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al.,“Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” GeneExpression Technology vol. 185 (1990), which is hereby incorporated byreference), and any derivatives thereof. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, mobilization, or electroporation. The DNA sequences arecloned into the vector using standard cloning procedures in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which ishereby incorporated by reference.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promotor whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eucaryotic promotorsdiffer from those of procaryotic promoters. Furthermore, eucaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference.

Promotors vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promotors in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promotors such as the T7 phage promoter, lac promotor, trppromotor, recA promotor, ribosomal RNA promotor, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promotor or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promotor unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promotor, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

Once the isolated DNA molecule encoding the fragment of a hypersensitiveresponse elicitor polypeptide or protein has been cloned into anexpression system, it is ready to be incorporated into a host cell. Suchincorporation can be carried out by the various forms of transformationnoted above, depending upon the vector/host cell system. Suitable hostcells include, but are not limited to, bacteria, virus, yeast, mammaliancells, insect, plant, and the like.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, and/or effecting insectcontrol for plants. These methods involve applying the fragment of ahypersensitive response elicitor polypeptide or protein, which fragmentitself elicits a hypersensitive response, in a non-infectious form toall or part of a plant or a plant seed under conditions effective forthe fragment to impart disease resistance, enhance growth, and/orcontrol insects. Alternatively, these fragments of a hypersensitiveresponse elicitor protein or polypeptide can be applied to plants suchthat seeds recovered from such plants themselves are able to impartdisease resistance in plants, to enhance plant growth, and/or to effectinsect control.

As an alternative to applying a fragment of a hypersensitive responseelicitor polypeptide or protein to plants or plant seeds in order toimpart disease resistance in plants, to effect plant growth, and/or tocontrol insects on the plants or plants grown from the seeds, transgenicplants or plant seeds can be utilized. When utilizing transgenic plants,this involves providing a transgenic plant transformed with a DNAmolecule encoding a fragment of a hypersensitive response elicitorpolypeptide or protein, which fragment elicits a hypersensitiveresponse, and growing the plant under conditions effective to permitthat DNA molecule to impart disease resistance to plants, to enhanceplant growth, and/or to control insects. Alternatively, a transgenicplant seed transformed with a DNA molecule encoding a fragment of ahypersensitive response elicitor polypeptide or protein which fragmentelicits a hypersensitive response can be provided and planted in soil. Aplant is then propagated from the planted seed under conditionseffective to permit that DNA molecule to impart disease resistance toplants, to enhance plant growth, and/or to control insects.

The embodiment of the present invention where the hypersensitiveresponse elicitor polypeptide or protein is applied to the plant orplant seed can be carried out in a number of ways, including: 1)application of an isolated fragment or 2) application of bacteria whichdo not cause disease and are transformed with a genes encoding thefragment. In the latter embodiment, the fragment can be applied toplants or plant seeds by applying bacteria containing the DNA moleculeencoding the fragment of the hypersensitive response elicitorpolypeptide or protein which fragment elicits a hypersensitive response.Such bacteria must be capable of secreting or exporting the fragment sothat the fragment can contact plant or plant seeds cells. In theseembodiments, the fragment is produced by the bacteria in planta or onseeds or just prior to introduction of the bacteria to the plants orplant seeds.

The methods of the present invention can be utilized to treat a widevariety of plants or their seeds to impart disease resistance, enhancegrowth, and/or control insects. Suitable plants include dicots andmonocots. More particularly, useful crop plants can include: alfalfa,rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweetpotato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout,beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach,onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin,zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape,raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.Examples of suitable ornamental plants are: Arabidopsis thaliana,Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation,and zinnia.

With regard to the use of the fragments of the hypersensitive responseelicitor protein or polypeptide of the present invention in impartingdisease resistance, absolute immunity against infection may not beconferred, but the severity of the disease is reduced and symptomdevelopment is delayed. Lesion number, lesion size, and extent ofsporulation of fungal pathogens are all decreased. This method ofimparting disease resistance has the potential for treating previouslyuntreatable diseases, treating diseases systemically which might not betreated separately due to cost, and avoiding the use of infectiousagents or environmentally harmful materials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: Pseudomonas solancearum,Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv.pelargonii. Plants can be made resistant, inter alia, to the followingfungi by use of the method of the present invention: Fusarium oxysporumand Phytophthora infestans.

With regard to the use of the fragments of the hypersensitive responseelicitor protein or polypeptide of the present invention to enhanceplant growth, various forms of plant growth enhancement or promotion canbe achieved. This can occur as early as when plant growth begins fromseeds or later in the life of a plant. For example, plant growthaccording to the present invention encompasses greater yield, increasedquantity of seeds produced, increased percentage of seeds germinated,increased plant size, greater biomass, more and bigger fruit, earlierfruit coloration, and earlier fruit and plant maturation. As a result,the present invention provides significant economic benefit to growers.For example, early germination and early maturation permit crops to begrown in areas where short growing seasons would otherwise precludetheir growth in that locale. Increased percentage of seed germinationresults in improved crop stands and more efficient seed use. Greateryield, increased size, and enhanced biomass production allow greaterrevenue generation from a given plot of land.

Another aspect of the present invention is directed to effecting anyform of insect control for plants. For example, insect control accordingto the present invention encompasses preventing insects from contactingplants to which the hypersensitive response elicitor has been applied,preventing direct insect damage to plants by feeding injury, causinginsects to depart from such plants, killing insects proximate to suchplants, interfering with insect larval feeding on such plants,preventing insects from colonizing host plants, preventing colonizinginsects from releasing phytotoxins, etc. The present invention alsoprevents subsequent disease damage to plants resulting from insectinfection.

The present invention is effective against a wide variety of insects.European corn borer is a major pest of corn (dent and sweet corn) butalso feeds on over 200 plant species including green, wax, and limabeans and edible soybeans, peppers, potato, and tomato plus many weedspecies. Additional insect larval feeding pests which damage a widevariety of vegetable crops include the following: beet armyworm, cabbagelooper, corn ear worm, fall armyworm, diamondback moth, cabbage rootmaggot, onion maggot, seed corn maggot, pickleworm (melonworm), peppermaggot, tomato pinworm, and maggots. Collectively, this group of insectpests represents the most economically important group of pests forvegetable production worldwide.

The method of the present invention involving application of thefragment of a hypersensitive response elicitor polypeptide or protein,which fragment elicits a hypersensitive response, can be carried outthrough a variety of procedures when all or part of the plant istreated, including leaves, stems, roots, etc. This may (but need not)involve infiltration of the hypersensitive response elicitor polypeptideor protein into the plant. Suitable application methods include high orlow pressure spraying, injection, and leaf abrasion proximate to whenelicitor application takes place. When treating plant seeds orpropagules (e.g., cuttings), in accordance with the applicationembodiment of the present invention, the fragment of the hypersensitiveresponse elicitor protein or polypeptide, in accordance with presentinvention, can be applied by low or high pressure spraying, coating,immersion, or injection. Other suitable application procedures can beenvisioned by those skilled in the art provided they are able to effectcontact of the fragment with cells of the plant or plant seed. Oncetreated with the fragment of the hypersensitive response elicitor of thepresent invention, the seeds can be planted in natural or artificialsoil and cultivated using conventional procedures to produce plants.After plants have been propagated from seeds treated in accordance withthe present invention, the plants may be treated with one or moreapplications of the fragment of the hypersensitive response elicitorprotein or polypeptide or whole elicitors to impart disease resistanceto plants, to enhance plant growth, and/or to control insects on theplants.

The fragment of the hypersensitive response elicitor polypeptide orprotein, in accordance with the present invention, can be applied toplants or plant seeds alone or in a mixture with other materials.Alternatively, the fragment can be applied separately to plants withother materials being applied at different times.

A composition suitable for treating plants or plant seeds in accordancewith the application embodiment of the present invention contains afragment of a hypersensitive response elicitor polypeptide or proteinwhich fragment elicits a hypersensitive response in a carrier. Suitablecarriers include water, aqueous solutions, slurries, or dry powders. Inthis embodiment, the composition contains greater than 500 nM of thefragment.

Although not required, this composition may contain additional additivesincluding fertilizer, insecticide, fungicide, nematacide, and mixturesthereof. Suitable fertilizers include (NH₄)₂NO₃. An example of asuitable insecticide is Malathion. Useful fungicides include Captan.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate the process of the present invention. In addition, thehypersensitive response eliciting fragment can be applied to plant seedswith other conventional seed formulation and treatment materials,including clays and polysaccharides.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a hypersensitive responseeliciting fragment need not be applied topically to the plants or seeds.Instead, transgenic plants transformed with a DNA molecule encoding sucha fragment are produced according to procedures well known in the art.

The vector described above can be microinjected directly into plantcells by use of micropipettes to transfer mechanically the recombinantDNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is herebyincorporated by reference. The genetic material may also be transferredinto the plant cell using polyethylene glycol. Krens, et al., Nature,296:72-74 (1982), which is hereby incorporated by reference.

Another approach to transforming plant cells with a gene which impartsresistance to pathogens is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes, or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference.

The DNA molecule may also be introduced into the plant cells byelectroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference. In this technique,plant protoplasts are electroporated in the presence of plasmidscontaining the expression cassette. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the plasmids. Electroporated plant protoplasts reform the cell wall,divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C.

Agrobacterium is a representative genus of the gram-negative familyRhizobiaceae. Its species are responsible for crown gall (A.tumefaciens) and hairy root disease (A. rhizogenes). The plant cells incrown gall tumors and hairy roots are induced to produce amino acidderivatives known as opines, which are catabolized only by the bacteria.The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome. J. Schell, Science, 237:1176-83 (1987), which is herebyincorporated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins. It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitingfragment resulting in disease resistance, enhanced plant growth, and/orcontrol of insects on the plant. Alternatively, transgenic seeds orpropagules (e.g., cuttings) are recovered from the transgenic plants.The seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants. The transgenicplants are propagated from the planted transgenic seeds under conditionseffective to impart disease resistance to plants, to enhance plantgrowth, and/or to control insects. While not wishing to be bound bytheory, such disease resistance, growth enhancement, and/or insectcontrol may be RNA mediated or may result from expression of thepolypeptide or protein fragment.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which ahypersensitive response eliciting fragment is applied. These othermaterials, including hypersensitive response eliciting fragments, can beapplied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the hypersensitive response eliciting fragment to impartdisease resistance, enhance growth, and/or control insects. Such plantsmay also be treated with conventional plant treatment agents (e.g.,insecticides, fertilizers, etc.).

EXAMPLES Example 1 Strains and Plasmids Used

The strains and plasmids used are set forth in Table 1 below

TABLE 1 E. amylovora Harpin, Plasmid source Brief Description, fragmentname strain Relevant Phenotype, Reference (or NA) pBCKS — Cm^(r)derivative of pBluescript KS. Stratagene, La Jolla, CA pBCSK — Cm^(r)derivative of pBluescript SK. Stratagene, La Jolla, CA pBSKS —pBluescript KS; Ap^(r). Stratagene, La Jolla, CA pBSSK II pBluescript IISK; Ap^(r). Stratagene, La Jolla, CA pBW7 — Mob + Tc^(r) helper plasmid.NA (Rella, et al., “Transposon Insertion Mutagenesis of Pseudomonasaeruginosa With a Tn5 Derivative: Application to Physical Mapping of thearc Gene Cluster,” Gene 33:293-303 (1985), which is hereby incorporatedby reference) pCPP51 — Broad host range derivative of NA pBSSK IIcontaining ori from pRO1614. PCPP430 321 Functional hrp gene cluster ofE. amylovora Ea321 cloned into pCPP9. Beer, S. V., et al., “The hrp GeneCluster of Erwinia Amylovora,” In Hennecke, H., and D. P. S. Verma,(eds.), Advances in Molecular Genetics of Plant-Microbe Interactions,Kluwer Academic Publishers, Dordrecht, Netherlands, 53-60 (1991), whichis hereby incorporated by reference. pCPP460 246 Functional hrp genecluster of E. amylovora Ea246 cloned into pCPP9. pCPP1104 321 1.2 kbPstI fragment of pCPP1084 Ea C31 in pBCKS pCPP1105 321 1.1 kb SmaIfragment of Ea C82 pCPP1084 in pBCSK pCPP1107 321 1.3 kb HindIIIfragment of Ea wt pCPP1084 in pBCSK pCPP1108 321 1.2 kb HincII-HindIIIof Ea N11 pCPP1084 in pBCSK pCPP1109 321 pCPP1107 with internal AvaII Ea1175 fragment deleted pCPP1110 321 As pCPP1108, but cloned into Ea N9pBCKS pCPP1111 321 367 bp TaqI fragment of Ea C305 pCPP1107 in pBCSKpCPP1113 246 As pCPP1109, but 425bp AvaII Ear 1175 fragment of pCPP1098deleted pCPP1119 246 Site specific mutation in Ear C368 pCPP1098; stopcodon inserted at L36 pCPP1120 246 Site specific mutation in Ear C281pCPP1098; stop codon inserted at T123 pCPP1121 321 702 bp KpnI fragmentinternal to Ea C375 hrpN deleted pCPP1127 246 3.1 kb BamH1 fragment ofEar wt pCPP1098 in pSU21 pCPP1128 246 Tn10 minikan in pCPP1127 Ear undefpCPP1136 246 4.4 kb EcoRI fragment of EAR pCPP1120, religated N122pCPP1146 246 4.2 kb EcoRI fragment of Ear N35 pCPP1119, religatedpCPP1147 321 1.2 kb BamH1 fragment of Ea wt pCPP1084, PCR amplified,cloned into pSU23 pCPP1148 246 As pCPp1147, but from pCPP1098 Ear wtpCPP1150 246 As pCPp1148, but in pCPP51 Ear wt vector pCPP1163 246 3.1kb BamH1 fragment of Ear wt pCPP1098 in pCPP51 pCPP1164 321 1.3 kbHindIII of pCPp1084 in Ea wt pCPP51 pCPP1165 Derivative of pCPP51w/KpnI, NA SacII sites removed pCPP1167 321 1.3 kb HindII fragment of Eawt pCPp1107 in pCPP1165 pCPP1169 246 As pCPP1167, but 3.1 kB BamHI Earwt insert from pCPP1098 pCPP1170 246 PCPP1098; Σ-Sp ligated into EarEcoRV site C128Σ pCPP1171 246 KpnI fragment internal to hrpN Ea C375deleted; shifted reading frame pCPP1172 321 Derivative of pCPP1167 within- Ea 1235 frame deletion of KpnI fragment internal to hrpN pCPP1173246 As pCPP1172, but from pCPP1169 Ear 1235 pCPP217 321 PCPP1084 with 2Styl fragments Ea C185 deleted, blunted, and religated pCPP1252 321PCPP1105 with Σ-Sp ligated at Ea C82Σ SmaI site pGPI-2 P15a ori.T7 RNApolymerase- N/A encoding plasmid; for protein expression (Tabor, S., etal., “A Bacteriophage T7 DNA Poly- merase/Promoter System for ControlledExclusive Expression of Specific Genes,” Proc. Natl. Acad. Sci. USA,82:1074-1078 (1985), which is hereby incorporated by reference). pHP45ΣAp^(r); Sp^(r); source of Ω-Sp fragment; N/A Ω (Fellay, R., et al.,“Interposon Mutagenesis of Soil and Water Bacteria a Family of DNAFragments Designed for in vitro Insertional Mutagenesis of Gram-Negative Bacteria,” Gene, 52:147- 154 (1987), which is herebyincorporated by reference). pSU21 P15a ori Km^(f)(Bartolomé, B. Y., N/Aet al., “Construction and N/A properties of a Family of pACYC184-DerivedCloning Vectors Compatible With pBR322 and its Derivatives,” Gene,102:75-78 (1991), which is hereby incorporated by reference). PSU23 P15aon Km^(f) (Bartolomé, B. Y., N/A et al., “Construction and properties ofa Family of pACYC184-Derived Cloning Vectors Compatible With pBR322 andits Derivatives,” Gene, 102:75- 78 (1991), which is hereby incorporatedby reference), Strains used E. amylovora Ea273Nx; Nalidixic acidresistant (Nx^(r)) derivative of Ea273. CUCPB 2348 E. amylovora Rifampinresistant derivative of Ea32. CUCPB 2545 E. coli GM272; dam-, dcm-,CUCPB 3047; (Blumenthal, R. M., et al., “E. coli Can Restrict MethylatedDNA and May Skew Genomic Libraries,” Trends in Biotech, 4:302-305(1986), which is hereby incorporated by reference) E. coli BL21(DE3);CUCPB 4277; (Studier, F. W., and B. A. Moffatt, “Use of Bacteriophage T7RNA Polymerase to Direct Selective High-level Expression of ClonedGenes,” J. Mol. Biol., 189:113-130 (1986), which is hereby incorporatedby reference) E. coli DH5α; (Nx^(r)). CUCPB 2475; Stratagene, La Jolla,CA.

Example 2 Molecular Biology Techniques

Several approaches were employed to obtain truncated or otherwisealtered versions of both E. amylovora harpins. These techniquesincluded: (i) subcloning of restriction fragments containing portions ofthe gene encoding the hypersensitive response elicitor protein orpolypeptide from Erwinia amylovora (i.e. hrpN) into expression vectors,by standard techniques (Sambrook, et al., Molecular Cloning: aLaboratory Manual, 2^(nd) ed. ed. Cold Spring Harbor, Laboratory,” ColdSpring Harbor, N.Y. (1989), which is hereby incorporated by reference);(ii) insertion of an Ω-fragment (Fellay, et al., “Interposon Mutagenesisof Soil and Water Bacteria a Family of DNA Fragments Designed for invitro Insertional Mutagenesis of Gram-Negative Bacteria,” Gene52:147-154 (1987), which is hereby incorporated by reference) into hrpN;(iii) site-specific mutagenesis approaches (Innis, et al., PCRProtocols. A Guide to Methods and Applications, Academic Press SanDiego, Calif. (1990); Kunkel, et al., “Rapid and Efficient Site-SpecificMutagenesis Without Phenotypic Selection,” Proc. Nat. Acad. Sci. USA82:488-492 (1985), which are hereby incorporated by reference); and (iv)creation of nested deletions (Erase-a-Base™ kit; Promega, Madison,Wis.). C-terminal deletion analysis of the hypersensitive responseelicitor protein or polypeptide from Erwinia amylovora (i.e.harpin_(Ea)) in pCPP1084 could not be performed because of the locationof restriction enzyme cleavage sites in pCPP1084. For N-terminaldeletions, pCPP1084 DNA was prepared using a Qiagen midiprep column(Qiagen, Chatsworth, Calif.) and digested with sst I followed by EcoRI.Subsequently, the digested DNA was subjected to exonuclease IIIdigestion, ligation, and transformation into E. coli BL21(DE3). Deletionsizes were estimated by agarose gel electrophoresis. Harpin fragmentswere named with respect to the portion of harpin deleted (e.g.,harpin_(Ea) C82 lacks the C-terminal 82 amino acid residues offull-length harpin_(Ea)).

Example 3 Protein Expression

For expression from T7 promoters, T7 RNA polymerase-dependent systemswere used. These systems utilized either strain E. coli BL21 (DE3)(Studier, et al., “Use of Bacteriophage T7 RNA Polymerase to DirectSelective High-Level Expression of Cloned Gene,” J. Mol. Biol.189:113-130 (1986), which is hereby incorporated by reference), orplasmid pGP1-2 (Tabor, et al., “A Bacteriophage T7 DNAPolymerase/Promoter System for Controlled Exclusive Expression ofSpecific Genes,”Proc. Natl. Acad. Sci., USA 82:1074-1078 (1985), whichis hereby incorporated by reference) in E. coli DH5α. Expression of hrpNfrom the T7 promoter was induced by addition of IPTG to a finalconcentration of 0.4 mM. For expression in E. amylovora Ea321 (i.e.harpin_(Ea)) or Ea273, pGP1-2 was introduced by transformation with a42° C. heat shock for 10 minutes, or by electroporation (Biorad GenePulser™). Hypersensitive response (i.e. HR)-eliciting activity wasscreened in tobacco cv. Xanthi leaves by in planta lysis (He, et al.,“Pseudomonas syringae pv. syringae harpin_(Pss): a Protein That isSecreted via the Hrp Pathway and Elicits the Hypersensitive Response inPlants,” Cell 73:1255-1266 (1993), which is hereby incorporated byreference) or by preparation of boiled and unboiled “CFEPs” (Wei, etal., “Harpin, Elicitor of the Hypersensitive Response Produced by thePlant Pathogen Erwinia amylovora,” Science 257:85-88 (1992), which ishereby incorporated by reference).

Example 4 In vitro Proteolysis of Harpin

In vitro proteolysis of harpin_(Ea) with Staphylococcus V8 proteinase(also termed endoproteinase Glu-C), trypsin, pepsin, and papain wasperformed as recommended (Scopes, et al., Protein Purification:Principles and Practice, 2^(nd) ed. Springer-Verlag. N.Y. (1987), whichis hereby incorporated by reference), for 2-16 hrs. at 20-37°.Endoproteinase Glu-C digestion was performed either in 50 mM ammoniumbicarbonate, pH 7.8 (in which cleavage occurs only after glutamic acid),or in 50 mM potassium phosphate, pH 7.8 (in which cleavage after bothglutamic acid and aspartic acid occurs).

Example 5 Plant-Derived Proteinases

Intercellular fluids (IF) were obtained from tobacco, tomato, apple,raspberry, and cotoneaster, as described (Hammond-Kosack, et al.,“Preparation and Analysis of Intercellular Fluid,” p. 15-21. In S. J.Gurr, M. J. McPherson, and D. J. Bowles (ed.), Molecular Plant PathologyA Practical Approach, 2^(nd) ed., The Practical Approach Series, IRLPublishers, Oxford (1992), which is hereby incorporated by reference),by vacuum infiltration of intercellular spaces with high-purity water.Proteolytic digestion of PAGE-purified harpin_(Ea) was performed for2-16 hrs. at 20-37° C., pH, by mixing equal volumes of IF withharpin_(Ea). A total leaf extract was obtained by grinding tobacco leafpanels with mortar and pestle in 5 mM potassium phosphate. The extractwas centrifuged and filtered, and the clarified ground leaf extract usedidentically as was the IF. Proteinase inhibitors were employed asfollows: Pepstatin A (final concentration 1 μM), E-64 (1 μM), Aprotinin(2μml), o-phenanthroline (1 mM), and p-mercuribenzoate (PCMB) (Sigma,St. Louis, Mo.).

Example 6 Peptide Purification

Peptide fragments of harpin obtained following digestion with tobacco IFwere fractionated by reverse-phase HPLC on a Vydac C 18 column using a2-60% acetonitrile gradient in 0.1% trifluoroacetic acid. Fractions werelyophilized, resuspended in 5 mM potassium phosphate and infiltratedinto tobacco leaf panels. The fraction with greatest HR-elicitingactivity was refractionated as above with a 35-70% acetonitrilegradient, and the purity of each fraction was assayed via gaschromatography-mass spectroscopy (GC-MS) and by N-terminal proteinsequencing at the Cornell Biotechnology Program Core Facility.

Example 7 Proteinase Activity-Stained Gels

Proteinase activity of IF was assayed in activity-stained polyacrylamidegels (Laemmli, “Cleavage of Structural Proteins During the Assembly ofthe Head of Bacteriophage T4,” Nature 227:680-685 (1970), which ishereby incorporated by reference) copolymerized with 0.1% gelatin(Heussen, et al., “Electrophoretic Analysis of Plasminogen Activators inPolyacrylamide Gels Containing Sodium Dodecyl Sulfate and CopolymerizedSubstrates,” Anal. Biochem. 102:196-202 (1980), which is herebyincorporated by reference). After electrophoresis, each gel was rinsedextensively to remove SDS and allow refolding of proteinases in the gel.Following additional incubation to allow proteolysis to occur, the gelswere stained with 0.1% Amido Black in 30% methanol/10% acetic acid. Eachgel stained darkly (due to the presence of copolymerized gelatin) exceptwhere proteinases had digested the gelatin, resulting in colorless bandsrepresenting the sites of proteinase activity.

Example 8 Truncated Harpins Retain HR-Eliciting Activity

The stability and the HR-eliciting activity of proteins encoded byvarious DNA constructs is shown in FIG. 1. Many DNA constructs encodingportions of harpin_(Ea) or harpin_(Ear) did not yield detectable proteinproducts following induction of expression in the T7 promoter-polymerasesystem (Tabor, et al., “A Bacteriophage T7 DNA Polymerase/PromoterSystem for Controlled Exclusive Expression of Specific Genes,”Proc.Natl. Acad. Sci. USA 82:1074-1078 (1985), which is hereby incorporatedby reference) and analysis of cell extracts by PAGE, possibly due toinstability of the encoded proteins. No DNA constructs (e.g., thoseobtained via Erase-a-Base™ protocol) yielded detectable protein productsdisplaying N-terminal deletions relative to the full-length protein. Nostable but inactive proteins were identified. Several constructsencoding proteins truncated at their C-terminus and often includingadditional vector-encoded amino acids yielded detectable products (e.g.harpin_(Ea) C82). In contrast, a construct encoding the same 321N-terminal amino acid residues of harpin_(Ea), but yielding a proteintruncated by the presence of an Ω-fragment (harpin_(Ea) C82 Ω) wasunstable (i.e. no product was detected). A construct encoding aharpin_(Ea) fragment with a large internal deletion (harpin_(Ea) I175)was also successfully used to express protein. These various truncatedproteins were tested for HR-eliciting activity. A 98 residue N-terminalharpin_(Ea) fragment (harpin_(Ea) C305) was the smallestbacterially-produced peptide that displayed HR-eliciting activity.

Example 9 Secretion of Harpin_(Ea) with an Altered C-Terminus

The effect of alteration at the harpin C-terminus on its secretion wasexamined. Harpin C32 contains the N-terminal 372 amino acids of harpin,but lacks the C-terminal 31 residues, which are replaced by 47 residuesencoded by the vector, resulting in a protein slightly larger than thewild type harpin_(Ea). The C31 protein retains HR-eliciting activity andis stable and easily expressed and detected by western analysis or PAGEbut it is no longer secreted into the culture supernatant as is the wildtype protein (FIG. 2). The presence of harpin_(Ea) C31 does notinterfere with secretion of the wild type harpin, which is found in boththe CFEP and the culture supernatant. However, harpin_(Ea) C31 is foundonly in the CFEP.

Example 10 Effect of Proteolysis on Harpin_(Ea)'s HR Eliciting Activity

In order to generate additional harpin_(Ea) fragments, purified fulllength protein was proteolyzed in vitro by several proteinases,including endoproteinase Glu-C, trypsin, pepsin, and papain (e.g., FIGS.3 and 4). Harpin solutions digested with trypsin or with papain lost allactivity. In contrast, following digestion with endoproteinase Glu-C,HR-eliciting activity was retained. No peptides larger than 6 kD wereevident by PAGE following trypsin digestion. Endoproteinase Glu-Cdigestion yielded an approximately 20 kD fragment, larger than expectedif all cleavage sites were cut, indicating that digestion was notcomplete (FIG. 4).

Example 11 Apoplastic Fluids (IF) Contain Harpin-Degrading ProteolyticActivity

Apoplastic fluids (intercellular fluids; IF) from tobacco and otherplants were also employed to proteolyze harpin. Each IF tested possessedproteinase activity(s), as indicated by the presence of multipleactivity-stained bands in polyacrylamide gels containing co-polymerizedgelatin (FIGS. 5A to 5C), as well as by the disappearance of detectableharpin_(Ea) (Schägger, et al., “Tricine-Sodium Dodecyl Sulfate GelElectrophoresis for the Separation of Proteins in the Range From 1 to100 kDa,” Anal. Biochem. 166:368-379 (1987), which is herebyincorporated by reference) following overnight digestion of purifiedharpin_(Ea) with IF. Proteinase activity was substantially greater at37° C. than at 20° C., and activity was higher at pH 8.5 than at pH 7.Several inhibitors were employed in order to define the proteolyticactivity(s) of the IF. No single proteinase inhibitor which was employedprevented degradation of harpin_(Ea). However, a mixture of theinhibitors Pepstatin A (1 μM), E-64 (1 μM), Aprotinin (2 μg/ml), ando-phenanthroline (1 mM), targeted at acid proteinases, cysteineproteinases, serine proteinases, and metalloproteinases, respectively,partially inhibited proteolysis.

Harpin_(Ea) degraded by proteolytic activities present in the plantapoplast retained HR-eliciting activity (FIG. 3). In contrast,harpin_(Ea) proteolyzed by a clarified extract produced by grindingtobacco leaf tissue with mortar and pestle lost HR-eliciting activity.In order to study whether apoplastic degradation of harpin was aprerequisite to its HR-eliciting activity, the length of time requiredfor leaf collapse when either intact harpin or harpin predigested withtobacco IF was infiltrated into tobacco leaf panels was compared. Bothpreparations elicited the HR in a similar time frame (12-18 hours,depending on the experiment).

Example 12 Characterization of HR-Eliciting Peptide Fragments

Peptides resulting from digestion by apoplastic plant proteinase(s) werefractionated by reverse phase HPLC (Vydac C18 column), and tested foractivity. Following treatment of intact harpin_(Ea) with tobacco IF,three fractions contained some HR-eliciting activity on tobacco. Two ofthe three demonstrated weak activity, and little protein was present.They were not further characterized. Fraction 19, which contained thestrongest activity as well as the most protein, was refractionated usinga more shallow elution gradient (FIG. 6). Refractionation, N-terminalprotein sequencing, and molecular weight analysis by mass spectroscopyindicated that four largely overlapping peptides were present. Peak 19-1contained peptides P91 and P95, corresponding to harpin_(Ea) residues110-200 and 110-204; peak 19-2 contained peptides P64 and P68,corresponding to harpin_(Ea) residues 137-200 and 137-204. 19-1 and 19-2each possessed HR-eliciting activity. The smallest peptide thusconfirmed to retain activity consisted of residues 137-204. The twopeptides in each peak were not separable under the conditions used.These active fragments are distinct from the smallest active N-terminalfragment (harpin_(Ea)C305), and indicate that more than one portion ofharpin_(Ea) displays activity in planta. Further digestion with trypsinabolished the HR-eliciting activity of 19-2. This proteinase cleaves P64and P68 as shown in FIG. 7. Further digestion with endoproteinase Glu-Cin ammonium bicarbonate buffer abolished the HR-eliciting activity of19-1. Endoproteinase Glu-C is predicted to cleave P91 and P95 as shownin FIG. 7. Loss of elicitor-activity followed further digestion of thesepeptides with endoproteinase Glu-C or trypsin.

Example 13 E. amylovora Harpin's Similarity with Other Proteins

The predicted protein sequences of proteinaceous HR elicitors fromseveral other bacterial plant pathogens, and of other proteins known tobe, or thought to be, secreted by a type III secretion pathway were alsocompared with that of harpin_(Ea). When harpin_(Ea) was compared withelicitors from E. amylovora Ea246 (i.e. harpin_(Ear)), Erwiniachrysanthemi EC 16 (harpin_(Ech)) (Bauer, et al., “Erwinia chrysanthemiharpin_(Ech): An Elicitor of the Hypersensitive Response ThatContributes to Soft-Rot Pathogenesis.”Mol. Plant-Microbe Interact8:484-491 (1995), which is hereby incorporated by reference), Erwiniacarotovora subsp. carotovora (harpin_(Ecc)) (Mukherjee, et al.,Presented at the 8^(th) International Congress Molecular Plant-MicrobeInteractions, Knoxville, Tenn. (1996), which is hereby incorporated byreference), Erwinia stewartii (Harpin_(Es)) (Frederick, et al., “The wtsWater-Soaking Genes of Erwinia stewartii are Related to hrp genes,”Presented at the Seventh International Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994), which is herebyincorporated by reference), Ralstonia (Pseudomonas) solanacearum (PopA)(Arlat, et al., “PopA1, a Protein Which Induces a Hypersensitivity-LikeResponse on Specific Petunia Genotypes, is Secreted via the Hrp Pathwayof Pseudomonas solanacearum,” EMBO J. 13:543-553 (1994), which is herebyincorporated by reference), Pseudomonas syringae 61 (harpin_(Pss)) (He,et al., “Pseudomonas syringae pv. syringae harpin_(Pss): a Protein Thatis Secreted via the Hrp Pathway and Elicits the Hypersensitive Responsein Plants,”Cell 73:1255-1266 (1993), which is hereby incorporated byreference), Pseudomonas syringae pv. tomato (harpin_(Pst)) (Preston, etal., “The HrpZ Proteins of Pseudomonas syringae pvs. syringae, glycinea,and tomato Are Encoded By An Operon Containing Yersinia ysc Homologs andElicit the Hypersensitive Response in Tomato But Not Soybean,” Mol.Plant-Microbe Interact 8:717-732 (1995), which is hereby incorporated byreference), the Erwinia-derived harpins contained significant regions ofsimilarity at the C-terminus. In addition, all the elicitors areglycine-rich, secreted, and heat-stable. Limited similarity betweenharpin_(Pss), and harpin_(Ea) had been reported previously (He, et al.,“Pseudomonas syringae pv. syringae harpin_(Pss): a Protein That isSecreted via the Hrp Pathway and Elicits the Hypersensitive Response inPlants,” Cell 73:1255-1266 (1993), which is hereby incorporated byreference), (Laby, et al., Presented at the Seventh InternationalSymposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland(1994), which is hereby incorporated by reference). A limited region ofsimilarity between harpin_(Ea) and other harpins frm Erwinia spp. wasalso evident at the extreme N-terminus of each protein, where 9 out ofthe first 26 residues are conserved (FIG. 8). Kyte-Doolittle hydropathyplots for each of the harpins produced by different Erwinia spp. areillustrated in FIG. 9. Each of the Erwinia harpins examined displays agenerally similar hydrophobicity profile along the full length of theprotein. This profile is distinct from the profile demonstrated by PopA1and by harpin_(Pss), and does not possess the symmetry evident in theprofile of those two proteins. The hydropathy profile of each Erwiniaharpin is generally similar to that of the others, yet distinct fromthat reported for harpin_(Pss) (Alfano, et al., “Analysis of the Role ofthe Pseudonionas Syringae HrpZ harpin in Elicitation of theHypersensitive Response to Tobacco Using Functionally Nonpolar hrpZDeletion Mutations, Truncated HrpZ Fragments, and hrmA Mutations,” Mol.Microbiol. 19:715-728 (1996), which is hereby incorporated byreference). Harpin_(Ecc) possesses a strikingly hydrophobic regionaround residues 54-143 (Mukherjee, et al., Presented at the 8^(th)International Congress Molecular Plant-Microbe Interactions, Knoxville,Tenn. (1996), which is hereby incorporated by reference). This portionof the protein is also the most hydrophobic region of harpin_(Ea) andharpin_(Es). The rest of each protein is predominantly hydrophilic.

Truncated proteins and fragments of harpin obtained followingproteolytic digestion of the full length protein indicate severalsurprising aspects of harpin_(Ea) HR-eliciting activity. These harpinfragments demonstrate that HR-eliciting activity resides in distinctregions of the protein, and that relatively small fragments of theprotein, as little as 68 residues and possibly less, are sufficient forthis activity. Fragments of other plant pathogen-derived elicitorproteins also retain biological activity, including Avr9 fromCaldosporium fulvum (Van den Ackervecken, et al., “The AVR9Race-Specific Elicitor of Cladosporium fulvum is Processed by Endogenousand Plant Proteases,” P1. Physiol. 103:91-96 (1993), which is herebyincorporated by reference), Pep-13 of Phytophthora megasperma(Nürnburger, et al., “High Affinity Binding of a Fungal OligopeptideElicitor to the Parsley Plasma Membranes Triggers Multiple DefenceResponses,” Cell, 78:449-460 (1994), which is hereby incorporated byreference), and harpin_(Pss) of P. syringae pv. syringae (Alfano, etal., “Analysis of the Role of the Pseudomonas syringae HrpZ harpin inElicitation of the Hypersensitive Response in Tobacco Using FunctionallyNonpolar hrpZ Deletion Mutations, Truncated HrpZ Fragments, and hrmAMutations,”Mol. Microbiol. 19:715-728 (1996), which is herebyincorporated by reference).

Expression of truncated harpin fragments and proteolysis of full-lengthharpins showed that two distinct fragments retain HR-eliciting activity.The primary sequence of each active fragment show no discernablesimilarity with each other, or with other elicitor-active peptides.However, the sites of cleavage by trypsin and endoproteinase Glu-Csuggest portions of each fragment required for activity. It would be ofinterest to alter specifically the amino acid residues at or near thesecleavage sites to determine whether HR-eliciting activity is altered orlost. Additionally, harpin_(Ea) P64 and P68 demonstrate distincthydrophobicity during reverse-phase HPLC (FIG. 6), and they correspondto a hydrophobic peak in a Kyte-Doolittle plot (FIG. 9). The role ofthis putative hydrophobic domain could be tested by mutagenesis, or bysynthesis of altered peptides. However, the fact that multiple fragmentsindependently possess HR-eliciting activity complicates analysis offull-length proteins.

This finding, that fragments of the protein retain HR-elicitingactivity, also allowed (at least) two apoplastic proteinase activities,which are distinct from intracellular plant proteinases, to be defined.Two apoplastic plant proteinases (from soybean) have been studied insome detail. SMEP, a metalloproteinase (Huangpu, et al., “Purificationand Developmental Analysis of an Extracellular Proteinase From YoungLeaves of Soybean,” Plant Physiol 108:969-974 (1995); McGeehan, et al.,“Sequencing and Characterization of the Soybean Leaf Metalloproteinase,”Plant Physiol. 99:1179-1183 (1992), which are hereby incorporated byreference) sensitive to EDTA, is thought to cleave at G/L and G/I.Interestingly, although there are 19 potential SMEP cleavage sites inthe intact harpin_(Ea), only one of them is located within fragments P91and P95, and none are within fragments P64 and P68 (FIG. 7). P91 and P95thus may represent partial digestion products of a SMEP-like proteinasein the tobacco apoplast. The other studies soybean apoplasticproteinase, SLAP, a sulfhydryl proteinase (Huangpu, et al.,“Purification and Developmental Analysis of an Extracellular ProteinaseFrom Young Leaves of Soybean,”Plant Physiol. 108:969-974 (1995), whichis hereby incorporated by reference) sensitive to p-chloromercuribenzoicacid (pCMB). Several lines of evidence suggest that multiple proteolyticactivities in the IF are degrading harpin_(Ea). PMSF, a serine proteaseinhibitor, decreases but does not entirely block harpin_(Ea) degradation(FIG. 5C); no single proteinase inhibitor tested blocks harpindegradation, and the cleavage sites after residues 109, 136, 200, and204 are dissimilar. Endoproteinase Glu-C does not abolish activity offull-length harpin, but does abolish activity of P91 and P95 (andpresumably P64 and P68); trypsin abolishes the activity of P64 and P68(and presumably P91 and P95). These final digests suggest specificportions of each distinct HR-eliciting peptide which are presumablynecessary for its activity, as mentioned previously.

The apoplastic activities degrade harpin without destroying itsHR-eliciting ability, in contrast to intracellular proteolyticactivities present in a ground leaf-extract, which abolish activity.This raises a number of intriguing questions, e.g., does the plant usethese harpin fragments as elicitor-signals? The timing of the HR wasexamined when full length harpin and harpin predigested by tobaccointercellular fluid were each infiltrated into tobacco leaves. The HRelicited by each preparation occurred 12-18 hours after infiltration.Co-infiltration of proteinase inhibitors into tobacco leaf panels alongwith harpin also had no effect on harpin's HR eliciting activity,although limited proteolytic degradation cannot be ruled out in thiscase, particularly since it seems that at least two, and perhaps more,apoplastic proteinases are present in tobacco. Because predigestedharpin elicited the HR no faster than undigested protein, proteolyticdigestion seems unlikely to be a rate-limiting step required for HR tooccur. The role of these apoplastic proteinases which are able tohydrolyze harpin partially, yet unable to abolish harpin's HR-elicitingactivity on tobacco, remains unclear. Salzer et al., “Rapid Reactions ofSpruce Cells to Elicitors Released From the Ectomycorrhizal FungusHebeloma crustuliniforme and Inactivation of These Elicitors byExtracellular Spruce Cell Enzymes,”Planta 198:118-126 (1996), which ishereby incorporated by reference, have noted that spruce (Picea abies(L.) Karst.) modulates the level of fungal cell wall elicitors releasedby the ectomycorrhizal fungus Hebeloma crustuliniforme by inactivatingthese molecules in the apoplast. They propose that Picea controls theelicitor level as part of its symbiotic interaction with the fungus.Similarly, PGIP of Phaseolus vulgaris has been suggested to modulate thelevel of elicitor-active oligogalacturonides present during theplant-parasite interaction in bean (Desiderio, et al.,“Polygalacturonase, PGIP, and Oligogalacturonides in Cell—CellCommunication,” Biochem. Sci. Trans. 22:394-397 (1994), which is herebyincorporated by reference). Perhaps the retention of HR-elicitingactivity by harpin fragments contributes to the ability of plants torecognize the presence of a pathogen. In this regard, it would beinteresting to explore whether transgenic host and non-host plants,engineered for apoplastic expression of a harpin activity-degradingproteinase, would exhibit reduced or increased sensitivity to E.amylovora, compared to non-engineered plants.

Despite numerous attempts, only a handful of truncated derivatives ofharpin_(Ea) and harpin_(Ear) were successfully expressed from portionsof hrpN. Problems with protein stability were evident in that severaltruncated harpins were unstable and difficult to purify. Additionally,expression of truncated harpins may be deleterious to bacteria.Truncated harpin_(Ea)C31 was, however, stable and easily purified, butnot secreted, suggesting that C-terminal sequences are involved inharpin secretion. Unfortunately, the presence of vector-encoded aminoacids in this protein complicates this conclusion. All attempts to cloneβ-galactosidase-harpin fusion proteins have been unsuccessful, as wereattempts to clone and express hrpN downstream of the lacZ promoter inseveral commonly used vectors such as pBluescript. Expression of suchconstructs is evidently deleterious to bacterial strains.

Wei, et al., “Harpin, Elicitor of the Hypersensitive Response ProducedBy the Plant Pathogen Erwinia amylovora,” Science, 257:85-88 (1992),which is hereby incorporated by reference, reported previously thatBLAST searches indicated harpin_(Ea), a possessed slight similarity toseveral other glycine-rich proteins, including keratins and glycine-richcell wall proteins. However, this is thought to be due to the highglycine content of harpin_(Ea), and as such does not suggest a role forharpin_(Ea). Examination of N-terminal sequences from severalHR-eliciting proteins produced by phytopathogenic bacteria (FIG. 8)yielded some potential similarities. However, the region in question isquite short. The region of putative primary sequence similarity islimited to the first 26 residues at the N-terminus, and its role remainsunclear. Surprisingly, E. carotovora harpin_(Ecc) appears more similarto the harpins from E. amylovora and E. stewartii than to that from E.chrysanthemi, to which it is more closely related with respect to itstaxonomic position as well as its mechanism of pathogenicity (i.e.soft-rots). In addition, although primary sequence similarity isstrongest only in the C-terminal third of each protein, the Erwiniaharpins possess broadly similar hydrophobicity profiles along theirentire lengths (FIG. 9). Based on its hydrophobicity profile, Alfano, etal., “Analysis of the Role of the Pseudomonas syringae HrpZ harpin inElicitation of the Hypersensitive Response in Tobacco Using FunctionallyNonpolar hrpZ Deletion Mutations, Truncated HrpZ Fragments, and hrmAMutations,” Mol. Microbiol. 19:715-728 (1996), which is herebyincorporated by reference, speculated that harpin_(Pss) may have anamphiphilic nature. However, the Erwinia harpins' profiles do not matchthat of harpin_(Pss).

Recently, a number of other secreted glycine-rich pathogenicityassociated proteins, elicitors of the HR or other plant-defenseresponses, have been described from other plant pathogenic bacteria andfungi (Boller, “Chemoperception of Microbial Signals in Plant Cells,”Ann. Rev. Plant Physiol. Plant Molec. Biol. 46:189-214 (1996), which ishereby incorporated by reference), including Phytophthora megasperma(Ballieul, et al., “A New Elicitor of the Hypersensitive Response inTobacco: a Fungal Glycoprotein Elicits Cell Death, Expression of DefenceGenes, Production of Salicylic Acid, and Induction of Systemic AcquiredResistance,” Plant Journal 8:551-560 (1995); Nürnburger, et al., “HighAffinity Binding of a Fungal Oligopeptide Elicitor to the Parsley PlasmaMembranes Triggers Multiple Defence Responses,” Cell 78:449-460 (1994),which are hereby incorporated by reference), and Magnaporthe grisea(Sweigard, et al., “Identification, Cloning, and Characterization ofPWL2, a Gene For Host Species Specificity in the Rice Blast Fungus,”Plant Cell 7:1221-1233 (1995), which is hereby incorporated byreference). Proteinaceous HR-elicitors have also now been described fromPhynchosporium secalis (Rohe, et al., “The Race-Specific Elicitor, NIP1,From the Barley Pathogen, Rhynchosporium secalis, Determines Avirulenceon Host Plants of the Rrs1 Resistance Genotype,” EMBO Journal14:4168-4177 (1995) which is hereby incorporated by reference, while P.infestans (Pieterse, et al., “Structure and Genomic Organization of theipiB and ipiO Gene Clusters of Phytophthora infestans,” Gene, 138:67-77(1994), which is hereby incorporated by reference) produces aglycine-rich pathogenicity-associated family of proteins of unknownfunction. Because the primary amino acid sequence of each elicitorprotein or peptide fragment shows no obvious similarity to that of theothers, it is unclear whether they interact with the same target on orin the plant cell, plasma membrane, or cell wall. In that regard, itmight be of interest to test whether any one of these molecules inhibitsthe action of other(s). The increasing availability of peptides such asPep13, Avr9, P68, and harpin_(Ea) C305 with plant-defenseresponse-eliciting activity (HR and otherwise) should enable preciseprobing of their targets on or in plant cells, as well as determinationof whether their mechanisms of activity are similar, distinct, oroverlapping.

Example 14 Bacterial Strains and Plasmids

Escherichia coli stains used in the following examples include DH5α andBL21(DE3) purchased from Gibco BRL and Stratagene, respectively. ThepET28(b) vector was purchased from Novagen. Eco DH5α/2139 contained thecomplete hrpN gene. The 2139 construct was produced by D. Bauer at theCornell University. The hrpN gene was cleaved from the 2139 plasmid byrestriction enzyme digestion with HindIII, then purified from an agarosegel to serve as the DNA template for PCR synthesis of truncated hrpNclones. These clones were subsequently inserted into the (His)₆ vectorpET28(b) which contained a Kan^(r) gene for selection of transformants.

Example 15 DNA Manipulation

Restriction enzymes were obtained from Boehringer Mannheim or Gibco BRL.T4 DNA ligase, Calf Intestinal Alkaline Phosphatase (CIAP), and PCRSupermix™ were obtained from Gibco BRL. The QIAprep Spin Miniprep Kit,the Qiagen Plasmid Mini Kit, and the QIAquick PCR Purification Kit werepurchased from Qiagen. The PCR primers were synthesized by LofstrandLabs Limited (Gaithersburg, Md.). The oligopeptides were synthesized byBio-Synthesis, Inc. (Lewisville, Tex.). All DNA manipulations such asplasmid isolation, restriction enzyme digestion, DNA ligation, and PCRwere performed according to standard techniques (molecular cloning) orprotocols provided by the manufacturer.

Example 16 Fragmentation of hrpN Gene

A series of N-terminal and C-terminal truncated hrpN genes and internalfragments were generated via PCR (FIG. 10). The full length hrpN genewas used as the DNA template and 3′ and 5′ primers were designed foreach truncated clone (FIG. 11). The 3′ primers contained in the NdeIenzyme cutting site which contained the start codon ATG (Methionine) andthe 5′ primers contained the stop codon TAA and a HindIII enzyme cuttingsite for ligation into the pET28(b) vector. PCR was carried out in 0.5ml tubes in a GeneAmp™ 9600 or 9700. 45 μl of Supermix™ were mixed with20 pmoles of each pair of DNA primers, 10 ng of full length harpin DNA,and diH₂O to a final volume of 50 μl. After heating the mixture at 95°C. for 2 min, the PCR was performed for 30 cycles at 94° C. for 1 min,58° C. for 1 min and 72° C. for 1.5 min. The PCR products were verifiedon a 6% TBE gel (Novex). Amplified DNA was purified with the QIAquickPCR purification kit, digested with Nde I and Hind III at 37° C. for 5hours, extracted once with phenol:chloroform:isoamylalcohol (25:25:1)and precipitated with ethanol. 5 μg of pET28(b) vector DNA were digestedwith 15 units of Nde I and 20 units of Hind III at 37° C. for 3 hoursfollowed with CIAP treatment to reduce the background resulting fromincomplete single enzyme digestion. Digested vector DNA was purifiedwith the QIAquick PCR purification kit and directly used for ligation.Ligation was carried out at 14-16° C. for 5-12 hours in a 15 μl mixturecontaining ca. 200 ng of digested pET28(b), 30 ng of targeted PCRfragment, and 1 unit T4 DNA ligase. 5-7.5 μl of ligation solution wereadded to 100 μl of DH5α competent cells in a 15 ml falcon tube andincubated on ice for 30 min. After a heat shock at 42° C. for 45seconds, 0.9 ml SOC solution or 0.45 ml LB media were added to each tubeand incubated at 37° C. for 1 hour. 20, 100, and 200 μl of transformedcells were placed onto LB agar with 30 μg/ml of kanamycin and incubatedat 37° C. overnight. Single colonies were transferred to 3 ml LB-mediaand incubated overnight at 37° C. Plasmid DNA was prepared from 2 ml ofculture with the QIAprep Miniprep kit. The DNA from the transformedcells was analyzed by restriction enzyme digestion or partial sequencingto verify the success of the transformations. Plasmids with the desiredDNA sequence were transferred into the BL21 strain using the standardchemical transformation method as indicated above. A clone containingthe full length harpin protein in the pet28(b) vector was generated as apositive control, and a clone with only the pET28(b) vector wasgenerated as a negative control.

Example 17 Expression of Harpin Truncated Proteins

Escherichia coli BL21(DE3) strains containing the hrpN clones were grownin Luria broth medium (g/L Difco Yeast extract, 10 g/L Difco Tryptone,5g/L NaCl, and 1 mM NaOH) containing 30 μg/ml of kanamycin at 37° C.overnight. The bacteria were then inoculated into 100 volumes of thesame medium and grown at 37° C. to an OD₆₂₀ of 0.6-0.8. The bacteriawere then inoculated into 250 volumes of the same medium and grown at37° C. to an OD₆₂₀ of ca. 0.3 or 0.6-0.8. One milli molar IPTG was thenadded and the cultures grown at 19° C. overnight (ca. 18 hours). Not allof the clones were successfully expressed using this strategy. Severalof the clones had to be grown in Terrific broth (12 g/L Bacto Tryptone,24 g/L Bacto yeast, 0.4% glycerol, 0.17 M KH₂PO₄, and 0.72 K₂HPO₄),and/or grown at 37° C. after IPTG induction, and/or harvested earlierthan overnight (Table 2).

TABLE 2 Expression of harpin truncated proteins amino acids (SEQ. ID.Fragment No. 23) Growth medium Induction O.D. Expression temp. Harvesttime  1  1-403 LB ca. 0.3 or 0.6- 19° C. or 25° C. 16-18 hr (+ control)0.8  2 — LB and TB ca. 0.3 or 0.6- 19° C. and 37° C. 16-18 hr (+control) 0.8  3 105-403 LB 0.6-0.8 19° C. 16-18 hr  4 169-403 TB ca. 0.319° C. 16-18 hr  5 210-403 LB or M9ZB 0.6-0.8 19° C. 16-18 hr  6 257-403LB or M9ZB 0.6-0.8 19° C. 16-18 hr  7 343-403 LB ca. 0.3 19° C. 5 hr  8 1-75 TB ca. 0.3 37° C. 16-18 hr  9  1-104 TB ca. 0.3 37° C. 16-18 hr 10 1-168 TB ca. 0.3 37° C. 16-18 hr 11  1-266 LB ca. 0.3 37° C. 4 hr 12 1-342 LB 0.6-0.8 19° C. 16-18 hr 13  76-209 LB ca. 0.3 37° C. 5 hr 14 76-168 TB or LB ca. 0.3 37° C. 3 hr or 16-18 hr 15 105-209 M9ZB ca. 0.337° C. 3 hr 16 169-209 no expression 17 105-168 LB ca. 0.3 37° C. 3-5 hr18  99-209 LB ca. 0.3 37° C. 3 hr 19 137-204 LB ca. 0.3 37° C. 3 hr 20137-180 LB ca. 0.3 37° C. 16-18 hr. 21 105-180 LB ca. 0.3 37° C. 3 hr 22150-209 no expression 23 150-180 no expression General expressionmethod: Escherichia coli BL21(DE3) strains containing the hrpN subcloneswere grown in Luria broth medium (5 g/L Difco Yeast extract, 10 g/LDifco Tryptone, 5 g/L NaCl, and 1 mM NaOH) containing 30 μg/ml ofkanamycin at 37° C. overnight. The bacteria were then inoculated into100 volumes of the same medium and grown at 37° C. to an OD₆₂₀ of0.6-0.8. The bacteria were then inoculated into 250 volumes of thegrowth # medium and grown at 37° C. to a specific induction OD₆₂₀. Onemilli molar IPTG was then added and the cultures grown at an optimaltemperature for protein expression, and harvested at a particular timefor recovery of the highest level of protein.

Example 18 Small Scale Purification of Harpin Truncated Proteins(Verification of Expression)

A 50 ml culture of a hrpN clone was grown as above to induce expressionof the truncated protein. Upon harvesting of the culture, 1.5 ml of thecell suspension were centrifuged at 14,000 rpm for 5 minutes,re-suspended in urea lysis buffer (8 M urea, 0.1 M Na₂HPO₄, and 0.01 MTris—pH 8.0), incubated at room temperature for 10 minutes, thencentrifuged again at 14,000 rpm for 10 minutes, and the supernatantsaved. A 50 μl aliquot of a 50% slurry of an equilibrated (His)₆-bindingnickel agarose resin was added to the supernatant and mixed at 4° C. forone hour. The nickel agarose was then washed three times with ureawashing buffer (8 M urea, 0.1 M Na₂HPO₄, and 0.01 M Tris—pH 6.3),centrifuging at 5,000 rpm for five minutes between washings. The proteinwas eluted from the resin with 50 μl of urea elution buffer (8 M urea,0.1 M Na₂HPO₄, 0.01 M Tris, and 0.1 MEDTA—pH 6.3). The eluate was run ona 4-20%, a 16%, or a 10-20% Tris-Glycine pre-cast gel depending upon thesize of the truncated protein to verify the expression.

Example 19 Induction of HR in Tobacco

A 1.5 ml aliquot from the 50 ml cultures grown for small scalepurification of the truncated proteins was centrifuged at 14,000 rpm forfour minutes and re-suspended in an equal volume of 5 mM potassiumphosphate buffer, pH 6.8. The cell suspension was sonicated for ca. 30seconds then diluted 1:2 and 1:10 with phosphate buffer. Both dilutionsplus the neat cell lysate were infiltrated into the fourth to ninthleaves of 10-15 leaf tobacco plants by making a hole in single leafpanes and infiltrating the bacterial lysate into the intercellular leafspace using a syringe without a needle. The HR response was recorded24-48 hr post infiltration. Tobacco (Nicotiana tabacun v. Xanthi)seedlings were grown in an environmental chamber at 20-25° C. with aphotoperiod of 12-h light/12-h dark and ca. 40% RH. Cell lysate was usedfor the initial HR assays (in order to screen the truncated proteins forHR activity) as the small scale urea purification yielded very littleprotein which was denatured due to the purification process.

Example 20 Large Scale Native Purification of Harpin Truncated Proteinsfor Comprehensive Biological Activity Assays

Six 500 ml cultures of a hrpN clone were grown as described earlier toinduce expression of the truncated protein. Upon harvesting of theculture the cells were centrifuged at 7,000 rpm for 5 minutes,re-suspended in imidazole lysis buffer (5 mM imidazole, 0.5 M NaCl, 20mM Tris) plus Triton X-100 at 0.05% and lysozyme at 0.1 mg/ml, andincubated at 30° C. for 15 minutes, sonicated for two minutes, thencentrifuged again at 15,000 rpm for 20 minutes, and the supernatant wassaved. A 4 ml aliquot of a 50% slurry of an equilibrated (His)₆-bindingnickel agarose resin was added to the supernatant and mixed at 4° C. forca. four hours. The nickel agarose was then washed three times withimidazole washing buffer (20 mM imidazole, 0.5 M NaCl, and 20 mM Tris),centrifuging at 5,000 rpm for five minutes between washings, then placedin a disposable chromatography column. The column was centrifuged at1100 rpm for one minute to remove any residual wash buffer and then theprotein was eluted from the resin with 4 ml of imidazole elution buffer(1 M imidazole, 0.5 M NaCl, and 20 mM Tris) by incubating the columnwith the elution buffer for ten minutes at room temperature and thencentrifuging the column at 1100 rpm for one minute. The eluate was runon a 4-20%, a 16%, or a 10-20% Tris-Glycine pre-cast gel depending uponthe size of the truncated protein to verify the expression. Theconcentration of the proteins was determined by comparison of theprotein bands with a standard protein in the Mark 12 molecular weightmarker.

Example 21 Large Scale Urea Purification of Harpin Truncated Proteinsfor Comprehensive Biological Activity Assay

The procedure was the same as the large scale native purification exceptthat urea lysis buffer, washing buffer, and elution buffer were used,and the cells were not sonicated as in the native purification. Afterpurification, the protein was renatured by dialyzing against lower andlower concentrations of urea over an eight hour period, then dialyzingovernight against 10 mM Tris/20 mM NaCl. The renaturing process causedthe N-terminal proteins to precipitate. The precipitated 1-168 proteinwas solubilized by the addition of 100 mM Tris-HCl at pH 10.4 thenheating the protein at 30° C. for ca. one hour. The concentration of theprotein was determined by comparison of the protein bands with astandard protein in the Mark 12 molecular weight marker. The 1-75 and1-104 protein fragments were not successfully solubilized using thisstrategy so they were sonicated in 100 mM Tris-HCl at pH 10.4 tosolubilize as much of the protein as possible and expose the activesites of the protein for the biological activity assays.

Example 22 Expression of Harpin Truncated Proteins

The small scale expression and purification of the fragment proteins wasdone to screen for expression and HR activity (Table 3).

TABLE 3 Expression and HR activity of harpin truncated proteins (smallscale screening) Amino Acids HR Fragment # (SEQ. ID. No. 23) Expressionactivity 1 (+ control)  1-403 + + 2 (− control) — background proteinonly −  3 105-403 + +  4 169-403 + −  5 210-403 + −  6 267-403 + −  7343-403 +/− −  8  1-75 + −  9  1-104 + +/− 10  1-168 + + 11  1-266 + +12  1-342 − + 13  76-209 + + 14  76-168 + − 15 105-209 + + 16 169-209 −− 17 105-168 + − 18  99-209 − + 19 137-204 + + 20 137-180 + + 21 105-180− + 22 150-209 − − 23 150-180 − −

All of the cloned fragment proteins were expressed to a certain degreeexcept for three small fragments (amino acids 169-209, 150-209, and150-180). The fragments were expressed at varying levels. Fragments210-403 and 267-403 were expressed very well, yielding a highconcentration of protein from a small scale purification, resulting in asubstantial protein band on SDS gel electrophoresis. Other fragments(such as a.a. 1-168 and 1-104) produced much less protein, resulting infaint protein bands upon electrophoresis. It was difficult to determinewhether fragment 343-403, the smallest C-terminal protein, wasexpressed, as there were several background proteins apparent in thegel, in addition to the suspected 343-403 protein. The positive andnegative control proteins consisting of the full length harpin proteinand only proteins, respectively, were tested for expression and HRactivity as well. large scale expression and purification of thefragment proteins was done the level of expression and titer of the HRactivity (Table 4).

TABLE 4 Expression level and HR titer of harpin truncated proteins(large sale purification) Amino acids Fragment # (SEQ. ID. No. 23)Expression HR titer 1 (+ control)  1-403 3.7 mg/ml 5-7 μg/ml 2 (−control) — — 1:2 dilution  4 169-403 2 mg/ml —  5 210-403 5 mg/ml —  6267-403 4 mg/ml —  7 343-402 200 μg/ml —  8  1-75 50 μg/ml —  9  1-10450 μg/ml 3 μg/ml (1:16 dilution) 10  1-168 1 mg/ml 1 μg/ml 13  76-2092.5 mg/ml 5 μg/ml 14  76-168 2 mg/ml — 15 105-209 5 mg/ml 5-10 μg/ml 17105-168 250 μg/ml — 19 137-204 3.6 mg/ml 3.5 μg/ml 20 137-180 250 μg/ml16 μg/ml

Not all of the proteins were expressed in large scale due to timeconstraints. The truncated proteins deemed to be the most important incharacterizing harpin were chosen. The positive control (full lengthharpin) was expressed in a relatively high level at 3.7 mg/ml. All ofthe C-terminal proteins were expressed at relatively high levels from2-5 mg/ml, except for fragment 343-403 as discussed earlier. TheN-terminal fragments were expressed very well also, however, during thepurification process, the protein precipitated and very little wasresolubilized. The concentrations in Table 3 reflect only thesolubilized protein. The internal fragments were expressed in the rangeof 2-3.6 mg/ml. It was extremely difficult to determine theconcentration of fragment 105-168 (it was suspected that theconcentration was much higher than indicated), as the protein bands onthe SDS gel were large, but poorly stained. The negative controlcontained several background proteins as expected, but no obviouslyinduced dominant protein.

Example 23 Induction of HR in Tobacco

The full length positive control protein elicited HR down to only5-7μg/ml. The negative control (pET 28) imidazole purified“protein”—which contained only background proteins—elicited an HRresponse down to the 1:2 dilution, which lowered the sensitivity of theassay as the 1:1 and 1:2 dilutions could not be used. This false HR waslikely due an affinity of the imidazole used in the purification processto bind to one or several of the background proteins, thereby notcompletely dialyzing out. Imidazole at a concentration of ca. 60 mM didelicit a false HR response.

One definitive domain encompassed a small internal region of the proteinfrom a.a. 137-180 (SEQ. ID. No. 23), a mere 44 a.a, is identified as thesmallest HR domain. The other potential HR domain is thought to belocated in the N-terminus of the protein from a.a. 1-104 (possibly a.a.1-75) (SEQ. ID. No. 23). It was difficult to confirm or narrow down theN-terminus HR domain due to the difficulties encountered in purifyingthese fragment proteins. The N-terminus fragment proteins had to bepurified with urea as no protein was recovered when the nativepurification process was used. Consequently, these proteins precipitatedduring the renaturing process and were difficult or nearly impossible toget back into solution, thereby making it hard to run the proteinsthrough the HR assay, as only soluble protein is able to elicit HR.Difficulty narrowing the N-terminus HR domain was only compounded by thefact that the negative control elicited false HR at the low dilutionlevels thereby reducing the sensitivity of the assay.

The internal domain proteins elicited an HR response between 5 and 10μg/ml of protein like the positive control, and the N-terminus domainproteins elicited an HR response between 1 and 3 μg/ml, lower than thepositive control.

Surprisingly, when the internal HR domain was cleaved between a.a. 168and 169 (fragments 76-168 and 105-168) (SEQ. ID. No. 23) the fragmentlost its HR activity. This suggests that the HR activity of fragment1-168 (SEQ. ID. No. 23) should not be attributed to the internal HRdomain, but rather to some other domain, leading to the assumption thatthere was likely a second HR domain to be found in the N-terminal regionof the protein. However, as discussed earlier it was difficult toconfirm this assumption.

The harpin C-terminus (a.a. 210-403 (SEQ. ID. No. 23)) did not containan HR domain. It did not elicit HR at a detectable level using thecurrent HR assay. Even the large C-terminal fragment from a.a. 169-403(SEQ. ID. No. 23) did not elicit HR even though it contained part of theinternal HR domain. As stated above, the protein between a.a. 168 and169 (SEQ. ID. No. 23) causes a loss of HR activity.

Because some of the small cloned proteins with 61 a.a. or less were notexpressed, several oligopeptides were synthesized with 30 a.a. to narrowdown the functional region of the internal HR domain. The oligopeptideswere synthesized within the range of a.a. 121-179 (SEQ. ID. No. 23).However, these oligos did not elicit the HR response. It was notexpected that there would be an HR response from oligos 137-166,121-150, and 137-156 (SEQ. ID. No. 23) as these fragments did notcontain the imperative amino acids 168 and 169 (SEQ. ID. No. 23). It wasexpected that the oligo 150-179 (SEQ. ID. No. 23) would elicit an HRresponse. It is possible that 30 a.a. is too small for the protein toelicit any activity due to a lack of folding and, therefore, a lack ofbinding or that during the synthesis of the peptides important aminoacids were missed (either in the process, or simply by the choice ofwhich 30 amino acids to synthesize) and, therefore, the fragments wouldnot be able to elicit HR. It is also possible, although unlikely, thatthese small proteins would have undergone some form ofpost-translational modification within the E. coli cell that they didnot contain when synthesized and, therefore, were not able to elicit anHR response.

Example 24 Biological Activity of HR Inducing Fragments

The two N-terminal harpin fragments spanning amino acids 1-104 and aminoacids 1-168 of the polypeptide of SEQ. ID. No. 23 were effective atinducing resistance of tobacco against TMV, in a similar manner as thefull length harpin protein. The internal fragments spanning amino acids76-209 and amino acids 105-209 of the polypeptide of SEQ. ID. No. 23were also effective at inducing TMV resistance. In addition, these samefour fragments conferred plant growth enhancement (“PGE”) in tomatoincreasing the height of the plants from 4-19% taller than the buffercontrol plants. The full length harpin protein induced growthenhancement of 6% greater than the buffer. The negative control did notinduce TMV resistance or growth enhancement.

TABLE 5 TMV resistance and PGE activity of HR inducing fragments derivedfrom harpin Amino acids HR TMV PGE ht > Fragment # (SEQ. ID. No. 23)activity resistance buffer 1 (+ control)  1-403 + +  6% 2 (− control) —− − −2%  9  1-104 + + 4-8% 10  1-168 + +  5-13% 13  76-209 + +  4-18% 15105-209 + +  6-19%

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 30(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 31 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGGAATTCAT ATGAGTCTGA ATACAAGTGG G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GGGAATTCAT ATGGGCGGTG GCTTAGGCGG T         #                  #          31 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GGCATATGTC GAACGCGCTG AACGATATG          #                  #            29 (2) INFORMATION FOR SEQ ID NO:4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GGGAATTCAT ATGTTAGGCG GTTCGCTGAA C         #                  #          31 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GGCATATGCT GAACACGCTG GGCTCGAAA          #                  #            29 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:GGCATATGTC AACGTCCCAA AACGACGAT          #                  #            29 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GGCATATGTC CACCTCAGAC TCCAGCG           #                  #             27 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GGGAATTCAT ATGCAAAGCC TGTTTGGTGA TGGG        #                  #        34 (2) INFORMATION FOR SEQ ID NO:9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GGGAATTCAT ATGGGTAATG GTCTGAGCAA G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GGGAATTCAT ATGAAAGCGG GCATTCAGGC G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GGGAATTCAT ATGACACCAG CCAGTATGGA GCAG        #                  #        34 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GCAAGCTTAA CAGCCCACCA CCGCCCATCA T         #                  #          31 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GCAAGCTTAA ATCGTTCAGC GCGTTCGACA G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GCAAGCTTAA TATCTCGCTG AACATCTTCA GCAG        #                  #        34 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GCAAGCTTAA GGTGCCATCT TGCCCATCAC          #                  #           30 (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:GCAAGCTTAA ATCAGTGACT CCTTTTTTAT AGGC        #                  #        34 (2) INFORMATION FOR SEQ ID NO:17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GCAAGCTTAA CAGGCCCGAC AGCGCATCAG T         #                  #          31 (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GCAAGCTTAA ACCGATACCG GTACCCACGG C         #                  #          31 (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GCAAGCTTAA TCCGTCGTCA TCTGGCTTGC TCAG        #                  #        34 (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:GCAAGCTTAA GCCGCGCCCA GCTTG           #                  #               25 (2) INFORMATION FOR SEQ ID NO:21:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 338 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:Met Gln Ile Thr Ile Lys Ala His Ile Gly Gl #y Asp Leu Gly Val Ser1               5    #                10   #                15Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu As #n Ser Ala Ala Ser Ser            20       #            25       #            30Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Th #r Ile Asp Lys Leu Thr        35           #        40           #        45Ser Ala Leu Thr Ser Met Met Phe Gly Gly Al #a Leu Ala Gln Gly Leu    50               #    55               #    60Gly Ala Ser Ser Lys Gly Leu Gly Met Ser As #n Gln Leu Gly Gln Ser65                   #70                   #75                   #80Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Le #u Leu Ser Val Pro Lys                85   #                90   #                95Ser Gly Gly Asp Ala Leu Ser Lys Met Phe As #p Lys Ala Leu Asp Asp            100       #           105       #           110Leu Leu Gly His Asp Thr Val Thr Lys Leu Th #r Asn Gln Ser Asn Gln        115           #       120           #       125Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Me #t Thr Gln Gly Asn Met    130               #   135               #   140Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Le #u Ser Ser Ile Leu Gly145                 1 #50                 1 #55                 1 #60Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Se #r Gln Pro Ser Leu Gly                165   #               170   #               175Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gl #y Ala Phe Asn Gln Leu            180       #           185       #           190Gly Asn Ala Ile Gly Met Gly Val Gly Gln As #n Ala Ala Leu Ser Ala        195           #       200           #       205Leu Ser Asn Val Ser Thr His Val Asp Gly As #n Asn Arg His Phe Val    210               #   215               #   220Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Il #e Gly Gln Phe Met Asp225                 2 #30                 2 #35                 2 #40Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Ty #r Gln Lys Asp Gly Trp                245   #               250   #               255Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Al #a Lys Ala Leu Ser Lys            260       #           265       #           270Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Me #t Asp Lys Phe Arg Gln        275           #       280           #       285Ala Met Gly Met Ile Lys Ser Ala Val Ala Gl #y Asp Thr Gly Asn Thr    290               #   295               #   300Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Se #r Leu Gly Ile Asp Ala305                 3 #10                 3 #15                 3 #20Ala Val Val Gly Asp Lys Ile Ala Asn Met Se #r Leu Gly Lys Leu Ala                325   #               330   #               335 Asn Ala(2) INFORMATION FOR SEQ ID NO:22:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 2141 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA CGGTATTCGA CA#CCGTTACG     60GCGTTTATGG CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA AT#CCGGCGTC    120GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA TGATGCAGAT TC#AGCCGGGG    180CAGCAATATC CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GG#CGGCAGAG    240TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GT#GGCCGCTG    300CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT GG#CGGGAATG    360ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC AGACAGGGAA CG#GACGCGCC    420CGATCATTAA GATAAAGGCG GCTTTTTTTA TTGCAAAACG GTAACGGTGA GG#AACCGTTT    480CACCGTCGGC GTCACTCAGT AACAAGTATC CATCATGATG CCTACATCGG GA#TCGGCGTG    540GGCATCCGTT GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AA#ATTATGCA    600AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GG#CTGGGTGC    660TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG TG#GATAAACT    720GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG ATGATGTTTG GC#GGCGCGCT    780GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG GCTGGGGATG AGCAATCAAC TG#GGCCAGTC    840TTTCGGCAAT GGCGCGCAGG GTGCGAGCAA CCTGCTATCC GTACCGAAAT CC#GGCGGCGA    900TGCGTTGTCA AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG AC#ACCGTGAC    960CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GC#CAGATGAC   1020CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT CC#ATTCTCGG   1080CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT TCTCTGGGGG CA#GGCGGCTT   1140GCAGGGCCTG AGCGGCGCGG GTGCATTCAA CCAGTTGGGT AATGCCATCG GC#ATGGGCGT   1200GGGGCAGAAT GCTGCGCTGA GTGCGTTGAG TAACGTCAGC ACCCACGTAG AC#GGTAACAA   1260CCGCCACTTT GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AG#TTTATGGA   1320TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GT#TCGCCGAA   1380GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG GT#ATGACCGG   1440CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC AAAAGCGCGG TG#GCGGGTGA   1500TACCGGCAAT ACCAACCTGA ACCTGCGTGG CGCGGGCGGT GCATCGCTGG GT#ATCGATGC   1560GGCTGTCGTC GGCGATAAAA TAGCCAACAT GTCGCTGGGT AAGCTGGCCA AC#GCCTGATA   1620ATCTGTGCTG GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GT#CTCTTTTC   1680TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TC#TGGTACAA   1740ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG CA#TCTTCCTC   1800GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA ATATAGAGAA AC#TCGCCGGC   1860CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC GC#CCCTTTAG   1920CAGATAGATT GCGGTTTCGT AATCAACATG GTAATGCGGT TCCGCCTGTG CG#CCGGCCGG   1980GATCACCACA ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AG#ATACCGAC   2040AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TC#TTGAGTTG   2100 GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T    #                   # 2141 (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 403 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Se #r Thr Met Gln Ile Ser1               5    #                10   #                15Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Le #u Gly Thr Ser Arg Gln            20       #            25       #            30Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gl #y Leu Gly Gly Gly Asn        35           #        40           #        45Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Le #u Leu Thr Gly Met Met    50               #    55               #    60Met Met Met Ser Met Met Gly Gly Gly Gly Le #u Met Gly Gly Gly Leu65                   #70                   #75                   #80Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Se #r Gly Gly Leu Gly Glu                85   #                90   #                95Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gl #y Gly Ser Leu Asn Thr            100       #           105       #           110Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Se #r Thr Thr Asn Ser Pro        115           #       120           #       125Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Se #r Gln Asn Asp Asp Ser    130               #   135               #   140Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Se #r Asp Pro Met Gln Gln145                 1 #50                 1 #55                 1 #60Leu Leu Lys Met Phe Ser Glu Ile Met Gln Se #r Leu Phe Gly Asp Gly                165   #               170   #               175Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gl #y Lys Gln Pro Thr Glu            180       #           185       #           190Gly Glu Gln Asn Ala Tyr Lys Lys Gly Val Th #r Asp Ala Leu Ser Gly        195           #       200           #       205Leu Met Gly Asn Gly Leu Ser Gln Leu Leu Gl #y Asn Gly Gly Leu Gly    210               #   215               #   220Gly Gly Gln Gly Gly Asn Ala Gly Thr Gly Le #u Asp Gly Ser Ser Leu225                 2 #30                 2 #35                 2 #40Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pr #o Val Asp Tyr Gln Gln                245   #               250   #               255Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Me #t Lys Ala Gly Ile Gln            260       #           265       #           270Ala Leu Asn Asp Ile Gly Thr His Arg His Se #r Ser Thr Arg Ser Phe        275           #       280           #       285Val Asn Lys Gly Asp Arg Ala Met Ala Lys Gl #u Ile Gly Gln Phe Met    290               #   295               #   300Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gl #n Tyr Gln Lys Gly Pro305                 3 #10                 3 #15                 3 #20Gly Gln Glu Val Lys Thr Asp Asp Lys Ser Tr #p Ala Lys Ala Leu Ser                325   #               330   #               335Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Se #r Met Glu Gln Phe Asn            340       #           345       #           350Lys Ala Lys Gly Met Ile Lys Arg Pro Met Al #a Gly Asp Thr Gly Asn        355           #       360           #       365Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Se #r Ser Leu Gly Ile Asp    370               #   375               #   380Ala Met Met Ala Gly Asp Ala Ile Asn Asn Me #t Ala Leu Gly Lys Leu385                 3 #90                 3 #95                 4 #00Gly Ala Ala (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1288 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TT#ATTCATAA     60GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT GC#AAATTTCT    120ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA GTCGCCAGAA TG#CTGGGTTG    180GGTGGCAATT CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CA#ATCAGCTG    240GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TG#GGCTGATG    300GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CC#TGGGCGAA    360GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GG#GCTCGAAA    420GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT GG#GTATTAAC    480TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT CCACCTCAGA CT#CCAGCGAC    540CCGATGCAGC AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TG#GTGATGGG    600CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CG#AGCAGAAC    660GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TC#TGAGCCAG    720CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GG#GTCTTGAC    780GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA CT#ACCAGCAG    840TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG GCATTCAGGC GC#TGAATGAT    900ATCGGTACGC ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TC#GGGCGATG    960GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GC#CGCAGTAC   1020CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AG#CACTGAGC   1080AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AG#CCAAGGGC   1140ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC AC#GCGGTGCC   1200GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG ATGCCATTAA CA#ATATGGCA   1260 CTTGGCAAGC TGGGCGCGGC TTAAGCTT         #                   #           1288 (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 341 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Le #u Gln Thr Pro Ala Met1               5    #                10   #                15Ala Leu Val Leu Val Arg Pro Glu Ala Glu Th #r Thr Gly Ser Thr Ser            20       #            25       #            30Ser Lys Ala Leu Gln Glu Val Val Val Lys Le #u Ala Glu Glu Leu Met        35           #        40           #        45Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Le #u Gly Lys Leu Leu Ala    50               #    55               #    60Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gl #y Gly Ile Glu Asp Val65                   #70                   #75                   #80Ile Ala Ala Leu Asp Lys Leu Ile His Glu Ly #s Leu Gly Asp Asn Phe                85   #                90   #                95Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gl #y Gln Gln Asp Leu Met            100       #           105       #           110Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Me #t Leu Asp Asp Leu Leu        115           #       120           #       125Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Gl #u Asp Asp Met Pro Met    130               #   135               #   140Leu Asn Lys Ile Ala Gln Phe Met Asp Asp As #n Pro Ala Gln Phe Pro145                 1 #50                 1 #55                 1 #60Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Le #u Lys Glu Asp Asn Phe                165   #               170   #               175Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Se #r Ala Leu Asp Ile Ile            180       #           185       #           190Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Al #a Gly Ser Leu Ala Gly        195           #       200           #       205Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Ph #e Ser Asn Asn Ser Ser    210               #   215               #   220Val Met Gly Asp Pro Leu Ile Asp Ala Asn Th #r Gly Pro Gly Asp Ser225                 2 #30                 2 #35                 2 #40Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Il #e Gly Glu Leu Ile Asp                245   #               250   #               255Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gl #y Leu Gly Thr Pro Val            260       #           265       #           270Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gl #y Gly Gln Ser Ala Gln        275           #       280           #       285Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Le #u Lys Gly Leu Glu Ala    290               #   295               #   300Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr As #p Val Gln Ser Ser Ala305                 3 #10                 3 #15                 3 #20Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Le #u Leu Gln Gly Thr Arg                325   #               330   #               335Asn Gln Ala Ala Ala             340 (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1026 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC CGGCAATGGC CC#TTGTCCTG     60GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA AGGCGCTTCA GG#AAGTTGTC    120GTGAAGCTGG CCGAGGAACT GATGCGCAAT GGTCAACTCG ACGACAGCTC GC#CATTGGGA    180AAACTGTTGG CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TG#AGGATGTC    240ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CG#CGTCTGCG    300GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA TG#GCCTGGCC    360AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG GGACAAGCTT CT#CCGAAGAC    420GATATGCCGA TGCTGAACAA GATCGCGCAG TTCATGGATG ACAATCCCGC AC#AGTTTCCC    480AAGCCGGACT CGGGCTCCTG GGTGAACGAA CTCAAGGAAG ACAACTTCCT TG#ATGGCGAC    540GAAACGGCTG CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TA#ATCAGCAG    600AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CA#GTTTTTCC    660AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CG#GTGACAGC    720GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC TTATCGACCG TG#GCCTGCAA    780TCGGTATTGG CCGGTGGTGG ACTGGGCACA CCCGTAAACA CCCCGCAGAC CG#GTACGTCG    840GCGAATGGCG GACAGTCCGC TCAGGATCTT GATCAGTTGC TGGGCGGCTT GC#TGCTCAAG    900GGCCTGGAGG CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GT#CGAGCGCT    960GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TC#AGGCTGCA   1020 GCCTGA                  #                  #                   #         1026 (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 344 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:Met Ser Val Gly Asn Ile Gln Ser Pro Ser As #n Leu Pro Gly Leu Gln1               5    #                10   #                15Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gl #n Gln Ser Gly Gln Ser            20       #            25       #            30Val Gln Asp Leu Ile Lys Gln Val Glu Lys As #p Ile Leu Asn Ile Ile        35           #        40           #        45Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Al #a Gly Gly Asn Thr Gly    50               #    55               #    60Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly As #n Ala Asn Ala Gly Ala65                   #70                   #75                   #80Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Se #r Gln Ala Pro Gln Ser                85   #                90   #                95Ala Asn Lys Thr Gly Asn Val Asp Asp Ala As #n Asn Gln Asp Pro Met            100       #           105       #           110Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Va #l Lys Leu Leu Lys Ala        115           #       120           #       125Ala Leu His Met Gln Gln Pro Gly Gly Asn As #p Lys Gly Asn Gly Val    130               #   135               #   140Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gl #y Gln Gly Gly Leu Ala145                 1 #50                 1 #55                 1 #60Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Al #a Gln Leu Gly Gly Gly                165   #               170   #               175Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Va #l Gly Gly Ala Gly Gly            180       #           185       #           190Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gl #y Gly Ala Asn Gly Ala        195           #       200           #       205Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Al #a Asn Gly Pro Gln Asn    210               #   215               #   220Ala Gly Asp Val Asn Gly Ala Asn Gly Ala As #p Asp Gly Ser Glu Asp225                 2 #30                 2 #35                 2 #40Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Le #u Met Lys Ile Leu Asn                245   #               250   #               255Ala Leu Val Gln Met Met Gln Gln Gly Gly Le #u Gly Gly Gly Asn Gln            260       #           265       #           270Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Al #a Ser Pro Ala Ser Gly        275           #       280           #       285Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Al #a Asp Asp Gln Ser Ser    290               #   295               #   300Gly Gln Asn Asn Leu Gln Ser Gln Ile Met As #p Val Val Lys Glu Val305                 3 #10                 3 #15                 3 #20Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gl #n Asn Gly Gly Ser Gln                325   #               330   #               335Gln Ser Thr Ser Thr Gln Pro Met             340(2) INFORMATION FOR SEQ ID NO:28:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 1035 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA CC#TGAACCTC     60AACACCAACA CCAACAGCCA GCAATCGGGC CAGTCCGTGC AAGACCTGAT CA#AGCAGGTC    120GAGAAGGACA TCCTCAACAT CATCGCAGCC CTCGTGCAGA AGGCCGCACA GT#CGGCGGGC    180GGCAACACCG GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CG#CGGGCGCC    240AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CA#ACAAGACC    300GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA GC#TGCTGGAA    360GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC AGCCCGGCGG CA#ATGACAAG    420GGCAACGGCG TGGGCGGTGC CAACGGCGCC AAGGGTGCCG GCGGCCAGGG CG#GCCTGGCC    480GAAGCGCTGC AGGAGATCGA GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TG#CTGGCGCC    540GGCGGCGCGG GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CG#GTGCGGGT    600 (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 26 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:Thr Leu Ile Glu Leu Met Ile Val Val Ala Il #e Ile Ala Ile Leu Ala1               5    #                10   #                15Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr             20      #            25 (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS:          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Gl #u Gln Gln Leu Asp Gln1               5    #                10   #                15Leu Leu Ala Met             20

What is claimed:
 1. An isolated fragment of a full length Erwiniaamylovora hypersensitive response elicitor polypeptide that ischaracterized by a high glycine content, no cysteine, and heatstability, wherein said fragment elicits a hypersensitive response inplants and is a fragment of the amino acid sequence of SEQ ID NO: 23spanning amino acids 105 and 403, 1 and 104, 1 and 168, 1 and 266, 1 and342, 1 and 321, 1 and 372, 76 and 209, 105 and 209, 99 and 209, 137 and204, 137 and 180, 105 and 180, 1 and 122, 1 and 98, 110 and 200, 110 and204, and 137 and
 200. 2. A method of imparting disease resistance toplants comprising: applying to a plant or a plant seed a fragment of afull length hypersensitive response elicitor polypeptide that ischaracterized by a high glycine content, no cysteine, and heatstability, which fragment elicits a hypersensitive response, is in anon-infectious form, and is a fragment of the amino acid sequence of SEQID NO: 23 spanning amino acids 105 and 403, 1 and 104, 1 and 168, 1 and266, 1 and 342, 1 and 321, 1 and 372, 76 and 209, 105 and 209, 99 and209, 137 and 204, 137 and 180, 105 and 180, 1 and 122, 1 and 98, 110 and200, 110 and 204, and 137 and 200, wherein said applying is carried outunder conditions effective to impart disease resistance.
 3. The methodaccording to claim 2, wherein plants are treated during said applying.4. The method according to claim 2, wherein plant seeds are treatedduring said applying, said method further comprising: planting the seedstreated with the fragment of the hypersensitive response elicitor innatural or artificial soil and propagating plants from the seeds plantedin the soil.
 5. A method of enhancing plant growth comprising: applyingto a plant or a plant seed a fragment of a full length hypersensitiveresponse elicitor polypeptide that is characterized by a high glycinecontent, no cysteine, and heat stability, which fragment elicits ahypersensitive response, is in a non-infectious form, and is a fragmentof the amino acid sequence of SEQ ID NO: 23 spanning amino acids 105 and403, 1 and 104, 1 and 168, 1 and 266, 1 and 342, 1 and 321, 1 and 372,76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 180, 105 and180, 1 and 122, 1 and 98, 110 and 200, 110 and 204, and 137 and 200,wherein said applying is carried out under conditions effective toenhance plant growth.
 6. The method according to claim 5, wherein plantsare treated during said applying.
 7. The method according to claim 5,wherein plant seeds are treated during said applying, said methodfurther comprising: planting the seeds treated with the fragment of thehypersensitive response elicitor in natural or artificial soil andpropagating plants from the seeds planted in the soil.
 8. The isolatedfragment according to claim 1, wherein the fragment spans amino acids105 and 403 of SEQ ID NO:
 23. 9. The isolated fragment according toclaim 1, wherein the fragment spans amino acids 1 and 104 of SEQ ID NO:23.
 10. The isolated fragment according to claim 1, wherein the fragmentspans amino acids 1 and 168 of SEQ ID NO:
 23. 11. The isolated fragmentaccording to claim 1, wherein the fragment spans amino acids 1 and 266of SEQ ID NO:
 23. 12. The isolated fragment according to claim 1,wherein the fragment spans amino acids 1 and 342 of SEQ ID NO:
 23. 13.The isolated fragment according to claim 1, wherein the fragment spansamino acids 1 and 372 of SEQ ID NO:
 23. 14. The isolated fragmentaccording to claim 1, wherein the fragment spans amino acids 76 and 209of SEQ ID NO:
 23. 15. The isolated fragment according to claim 1,wherein the fragment spans amino acids 105 and 209 of SEQ ID NO:
 23. 16.The isolated fragment according to claim 1, wherein the fragment spansamino acids 99 and 209 of SEQ ID NO:
 23. 17. The isolated fragmentaccording to claim 1, wherein the fragment spans amino acids 137 and 204of SEQ ID NO:
 23. 18. The isolated fragment according to claim 1,wherein the fragment spans amino acids 137 and 180 of SEQ ID NO:
 23. 19.The isolated fragment according to claim 1, wherein the fragment spansamino acids 105 and 180 of SEQ ID NO:
 23. 20. The isolated fragmentaccording to claim 1, wherein the fragment spans amino acids 1 and 122of SEQ ID NO:
 23. 21. The isolated fragment according to claim 1,whereinthe fragment spans amino acids 1 and 98 of SEQ ID NO:
 23. 22. Theisolated fragment according to claim 1, wherein the fragment spans aminoacids 110 and 200 of SEQ ID NO:
 23. 23. The isolated fragment accordingto claim 1, wherein the fragment spans amino acids 110 and 204 of SEQ IDNO:
 23. 24. The isolated fragment according to claim 1, wherein thefragment spans amino acids 137 and 200 of SEQ ID NO:
 23. 25. Theisolated fragment according to claim 1, wherein the fragment spans aminoacids 1 and 321 of SEQ ID NO:
 23. 9
 26. The method according to claim 2,wherein the fragment spans amino acids 105 and 403 of SEQ ID NO:
 23. 27.The method according to claim 2, wherein the fragment spans amino acids1 and 104 of SEQ TD NO:
 23. 28. The method according to claim 2, whereinthe fragment spans amino acids 1 and 168 of SEQ ID NO:
 23. 29. Themethod according to claim 2, wherein the fragment spans amino acids 1and 266 of SEQ ID NO:
 23. 30. The method according to claim 2, whereinthe fragment spans amino acids 1 and 342 of SEQ ID NO:
 23. 31. Themethod according to claim 2, wherein the fragment spans amino acids 1and 372 of SEQ ID NO:
 23. 32. The method according to claim 2, whereinthe fragment spans amino acids 76 and 209 of SEQ ID NO:
 23. 33. Themethod according to claim 2, wherein the fragment spans amino acids 105and 209 of SEQ ID NO:
 23. 34. The method according to claim 2, whereinthe fragment spa acids 99 and 209 of SEQ ID NO:
 23. 35. The methodaccording to claim 2, wherein the fragment spans amino acids 137 and 204of SEQ ID NO:
 23. 36. The method according to claim 2, wherein thefragment spans amino acids 137 and 180 of SEQ ID NO:
 23. 37. The methodaccording to claim 2, wherein the fragment spans amino acids 105 and 180of SEQ ID NO:
 23. 38. The method according to claim 2, wherein thefragment spans amino acids 1 and 122 of SEQ ID NO:
 23. 39. The methodaccording to claim 2, wherein the fragment spans amino acids 1 and 98 ofSEQ ID NO:
 23. 40. The method according to claim 2, wherein the fragmentspan amino acids 110 and 200 of SEQ ID NO:
 23. 41. The method accordingto claim 2, wherein the fragment spans amino acids 110 and 204 of SEQ IDNO:
 23. 42. The method according to claim 2, wherein the fragment spansamino acids 137 and 200 of SEQ ID NO:
 23. 43. The method according toclaim 2, wherein the fragment spans amino acids 1 and 321 of SEQ ID NO:23.
 44. The method according to claim 5, wherein the fragment spansamino acids 105 and 403 of SEQ ID NO:
 23. 45. The method according toclaim 5, wherein the fragment spans amino acids 1 and 104 of SEQ ID NO:23.
 46. The method according to claim 5, wherein the fragment spansamino acids 1 and 168 of SEQ ID NO:
 23. 47. The method according toclaim 5, wherein the fragment spans amino acids 1 and 266 of SEQ ID NO:23.
 48. The method according to claim 5, wherein the fragment spansamino acids 1 and 342 of SEQ ID NO:
 23. 49. The method according toclaim 5, wherein the fragment spans amino acids 1 and 372 of SEQ ID NO:23.
 50. The method according to claim 5, wherein the fragment spansamino acids 76 and 209 of SEQ ID NO:
 23. 51. The method according toclaim 5, wherein the fragment spans amino acids 105 and 209 of SEQ IDNO:
 23. 52. The method according to claim 5, wherein the fragment spansamino acids 99 and 209 of SEQ ID NO:
 23. 53. The method according toclaim 5, wherein the fragment spans amino acids 137 and 204 of SEQ IDNO:
 23. 54. The method according to claim 5, wherein the fragment spansamino acids 137 and 180 of SEQ ID NO:
 23. 55. The method according toclaim 5, wherein the fragment spans amino acids 105 and 180 of SEQ IDNO:
 23. 56. The method according to claim 5, wherein the fragment spansamino acids 1 and 122 of SEQ ID NO:
 23. 57. The method according toclaim 5, wherein the fragment spans amino acids 1 and 98 of SEQ ID NO:23.
 58. The method according to claim 5, wherein the fragment spansamino acids 110 and 200 of SEQ ID NO:
 23. 59. The method according toclaim 5, wherein the fragment spans amino acids 110 and 204 of SEQ IDNO:
 23. 60. The method according to claim 5, wherein the fragment spanamino acids 137 and 200 of SEQ ID NO:
 23. 61. The method according toclaim 5, wherein the fragment spans amino acids 1 and 321 of SEQ ID NO:23.